Antibodies to CD70

ABSTRACT

The present invention relates to antibodies and antigen binding fragments thereof which bind to the human CD70 protein with high affinity and display potent inhibition of tumor cell growth.

TECHNICAL FIELD

The present invention relates to antibodies and antigen bindingfragments thereof which bind to the human CD70 protein with highaffinity and display potent inhibition of tumour cell growth.

BACKGROUND

The cytokine receptor CD27 is a member of the tumour necrosis factorreceptor (TFNR) superfamily, which play a role in cell growth anddifferentiation, as well as apoptosis. The ligand for CD27 is CD70,which belongs to the tumour necrosis factor family of ligands. CD70 is a193 amino acid polypeptide having a 20 amino acid hydrophilic N-terminaldomain and a C-terminal domain containing 2 potential N-linkedglycosylation sites (Goodwin, R. G. et al. (1993) Cell 73:447-56; Bowmanet al. (1994) Immunol 152: 1756-61). Based on these features, CD70 wasdetermined to be a type II transmembrane protein having an extracellularC-terminal portion.

CD70 is transiently found on activated T and B lymphocytes and dendriticcells (Hintzen et al. (1994) J. Immunol. 152: 1762-1773; Oshima et al.(1998) Int. Immunol. 10:517-26; Tesselaar et al. (2003) J. Immunol.170:33-40). In addition to expression on normal cells, CD70 expressionhas been reported in different types of cancers including renal cellcarcinomas, metastatic breast cancers, brain tumours, leukemias,lymphomas and nasopharyngeal carcinomas (Junker et al. (2005) J Urol.173:2150-3; Sloan et al. (2004) Am J Pathol. 164:315-23; Held-Feindt andMentlein (2002) Int J Cancer 98:352-6; Hishima et al. (2000) Am J SurgPathol. 24:742-6; Lens et al. (1999) Br J Haematol. 106:491-503). Theinteraction of CD70 with CD27 has also been proposed to play a role incell-mediated autoimmune disease and the inhibition of TNF-alphaproduction (Nakajima et al. (2000) J. Neuroimmunol. 109:188-96).

Accordingly, CD70 represents a target for the treatment of cancer,autoimmune disorders and a variety of other diseases characterized byCD70 expression.

WO 2006/0044643 describes CD70 antibodies containing an antibodyeffector domain which can mediate one or more of ADCC, ADCP, CDC or ADCand either exert a cytostatic or cytotoxic effect on a CD70-expressingcancer or exert an immunosuppressive effect on a CD70-expressingimmunological disorder in the absence of conjugation to a cytostatic orcytotoxic agent. The antibodies exemplified therein are based on theantigen-binding regions of two monoclonal antibodies, denoted 1F6 and2F2.

WO 2007/038637 describes fully human monoclonal antibodies that bind toCD70. These antibodies are characterised by binding to human CD70 with aK_(D) of 1×10⁻⁷ M or less. The antibodies also bind to, and areinternalised by, renal cell carcinoma tumor cell lines which expressCD70, such as 786-O.

SUMMARY OF THE INVENTION

Provided herein are antibodies, or antigen binding fragments thereof,(referred to herein as CD70 antibodies) which bind to the human CD70protein and exhibit properties which are different, and generallysuperior, to CD70 antibodies described in the prior art. The superiorproperties of these antibodies are advantageous with regard to use inhuman therapy, particularly treatment of CD70-expressing cancers andalso immunological disorders.

The CD70 antibodies described herein are characterised by extremely highbinding affinity for human CD70. All preferred embodiments describedherein exhibit a binding affinity for recombinant human CD70 (measuredby Biacore™ surface plasmon resonance as described herein) which issignificantly higher than the most potent prior art CD70 antibodiesproposed for human therapy, including prior art CD70 antibodies of“fully human” origin. In addition, all preferred embodiments of the CD70antibodies described herein exhibit superior (i.e. higher affinity)binding to CD70 expressed on the surface of human cell lines,specifically human cancer cell lines, when compared to prior art CD70antibodies proposed for human therapy. This superior binding tocell-surface CD70 is particularly marked in regard to human cancer celllines which express CD70 at “low copy number” and is of direct relevanceto use of the antibodies in human therapy. Still further, preferredembodiments of the CD70 antibodies described herein exhibitsubstantially improved binding to cancer cells isolated from humanpatients, particularly cancer cells isolated from patients with chroniclymphocytic leukaemia (CLL), when compared to prior art CD70 antibodiesproposed for human therapeutic use.

Therefore, in a first aspect of the invention there is provided anantibody, or an antigen binding fragment thereof, which binds to humanCD70, said antibody or antigen binding fragment comprising at least oneheavy chain variable domain (VH) and at least one light chain variabledomain (VL), wherein said VH and VL domain exhibit an off-rate (k_(off)measured by Biacore™) for human CD70 of less than 7×10⁴ s⁻¹, when testedas a Fab fragment using the standard Biacore™ protocol described herein.

In a preferred embodiment the antibody or antigen binding fragmentcomprises at least one heavy chain variable domain (VH) and at least onelight chain variable domain (VL), wherein said VH and VL domain exhibitan off-rate for human CD70 of 5×10⁻⁴ s⁻¹ or less, or 2×10⁻⁴ s⁻¹ or less,or 1×10⁻⁴ s⁻¹ or less. Most preferably the CD70 antibody will exhibit anoff-rate for CD70 in the range of from 0.4×10⁻⁴ s⁻¹ to 4.8×10⁻⁴ s⁻¹,when tested as a Fab fragment.

There is also provided an antibody which binds to human CD70, saidantibody comprising two Fab regions, wherein each of the Fab regionsbinds to human CD70 and exhibits an off-rate for human CD70 of 5×10⁻⁴s⁻¹ or less, or 2×10⁻⁴ s⁻¹ or less, or 1×10⁻⁴ s⁻¹ or less and preferablyin the range of from 0.4×10⁻⁴ s⁻¹ to 4.8×10⁻⁴ s⁻¹, when tested as a Fabfragment. The two Fab regions may be identical or they may differ interms of binding properties, e.g. affinity for human CD70. The two Fabregions may bind to the same epitope or overlapping epitopes on humanCD70 or they may bind to distinct, non-overlapping epitopes on humanCD70. The two Fab regions may differ from one another in terms of aminoacid sequence within one or both of the VH and VL domains.

Preferred embodiments of the CD70 antibodies provided herein may, inaddition to the extremely high binding affinity for CD70, exhibit potentblocking or inhibition of the interaction between CD70 and its ligandCD27. The preferred CD70 antibodies which exhibit both high affinitybinding to CD70 and potent blocking of the CD70/CD27 interaction areparticularly advantageous as therapeutic agents for treatment of diseaseindications where blocking of CD70/CD27 signalling enhances therapeuticefficacy (e.g. in addition to cell-killing mediated by the effectorfunctions of the CD70 antibody), for example autoimmune diseases andcancers which co-express CD70 and CD27.

Not all of the CD70 antibodies described herein exhibit potent blockingof the CD70/CD27 interaction in addition to the high affinity binding toCD70. Also described herein are a number of CD70 antibodies whichexhibit very high affinity binding to CD70 but do not show significantblocking of the CD70/CD27 interaction. The properties of theseantibodies are described elsewhere herein. The availability of highaffinity non-blocking CD70 antibodies may enhance/extend the range oftherapeutic possibilities.

Preferred embodiments of the CD70 antibodies described herein,exhibiting very high binding affinity for human CD70, are alsocharacterised by a combination of binding properties which is notexhibited by prior art CD70 antibodies proposed for human therapeuticuse. Accordingly, the preferred CD70 antibodies described herein arecharacterised by:

-   -   (a) binding within the amino acid sequence HIQVTLAICSS (SEQ ID        NO:342) in human CD70;    -   (b) cross-reactivity with CD70 homologs of rhesus macaque        (Macaca mulatta) and cynomolgus monkey (Macaca cynomolgus);    -   (c) binding to both native human CD70 and heat denatured        recombinant human CD70.

This combination of binding properties, which is not exhibited by theprior art antibodies proposed for human therapeutic use, indicatesbinding to a novel epitope on CD70 which is different to the epitopesbound by prior art CD70 antibodies.

The combination of binding properties exhibited by the preferred CD70antibodies is advantageous in the context of human drug development. Inparticular, cross-reactivity with simian CD70 homologs enablestoxicology studies on CD70 antibodies proposed for human therapeutic useto be carried out in primate models.

The preferred CD70 antibodies described herein still further exhibitfavourable properties which are relevant to commercial manufacture as atherapeutic antibody product. As discussed elsewhere herein, thepreferred CD70 antibodies provided herein exhibit extremely high levelexpression in the recombinant expression systems utilised for commercialmanufacture of clinical grade therapeutic antibody products. Theexpression levels achievable with the preferred CD70 antibodies farexceed the levels typically achieved even for “fully human” therapeuticantibody products. In addition, the preferred CD70 antibody products(produced by recombinant expression in a format suitable for humantherapeutic use) exhibit outstanding thermal stability, which issuperior to typical therapeutic antibody products.

The CD70 antibodies provided herein with superior binding affinity forhuman CD70 and the other advantageous properties listed above arecamelid-derived (for example llama-derived). The camelid-derived CD70antibodies may be isolated or recombinantly expressed monoclonalantibodies. Preferred embodiments may be a humanised (or germlined)monoclonal antibody (e.g. a humanised variant of a camelid-derivedantibody), a chimeric antibody (e.g. a camelid-human chimeric antibody)or a humanised chimeric antibody (e.g. a chimeric antibody comprisinghumanised variants of camelid VH and VL domains and constant domains ofa human antibody).

Camelid-derived CD70 antibodies may comprise at least one hypervariableloop or complementarity determining region obtained from a VH domain ora VL domain of a species in the family Camelidae. In a particularembodiment, the CD70 antibody, or antigen binding fragment thereof, maycomprise a heavy chain variable domain (VH) and light chain variabledomain (VL), wherein the VH and VL domains, or one or more CDRs thereof,are camelid-derived. In particular embodiments the antibody or antigenbinding fragment thereof may comprise llama VH and VL domains, or humangermlined variants of llama VH and VL domains. This antibody, or antigenbinding fragment, may exhibit “high human homology”, as defined herein.

The camelid-derived CD70 antibodies described herein typically exhibitVH and/or VL region amino acid sequences having at least 90% (e.g., 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity the closestmatching human antibody germline sequence.

Further preferred embodiments of the invention include humanised (orhuman germlined) variants of the camelid-derived CD70 antibodies. Inparticular, the invention provides humanised or human germlined variantsof the llama-derived CD70 antibodies described herein.

In a further aspect of invention there is provided a chimericcamelid-human antibody which binds human CD70, wherein theantigen-binding portions of the antibody (e.g. VH and/or VL domains orCDRs thereof) are camelid-derived and the constant regions of theantibody are derived from a human antibody. In particular, the inventionprovides a chimeric llama-human antibody which binds human CD70.

In a further aspect of invention there is provided a humanised variantof a chimeric camelid-human antibody which binds human CD70, wherein theantigen-binding portions of the antibody (e.g. VH and/or VL domains orCDRs thereof) are humanised variants of camelid-derived sequences andthe constant regions of the antibody are derived from a human antibody.In particular, the invention provides a humanised variant of a chimericllama-human antibody which binds human CD70.

Preferred (but non-limiting) embodiments of the CD70 antibodies, orantigen binding fragments thereof, are defined below by reference tospecific structural characteristics, i.e. specified amino acid sequencesof either the CDRs (one or more of SEQ ID Nos: 49-59, 262 or 263 (heavychain CDR3), or SEQ ID Nos: 26-37, 249, 258 or 259 (heavy chain CDR2) orSEQ ID Nos: 10-20, 248, 256 or 257 (heavy chain CDR1) or one of the CDRsequences shown as SEQ ID NOs: 148-168, 271 or 273 (light chain CDR3),or SEQ ID Nos: 109-128 or 270 (light chain CDR2) or SEQ ID Nos:77-95, or250-253, 267 or 268 (light chain CDR1), or entire variable domains (oneor more of SEQ ID NOs: 177-188, 212-223, 274 or 275 (VH) or SEQ IDNos:189-211, 230-245, 276 or 277 (VL)). All of these antibodies bind tohuman CD70 with high affinity, exhibiting an off-rate for human CD70 of5×10⁻⁴ s⁻¹ or less, and typically in the range of from 0.4×10⁻⁴ s⁻¹ to4.8×10⁻⁴ s⁻¹, when tested as a Fab fragment.

The invention also provides humanised/germlined variants of theseantibodies, plus affinity variants and variants containing conservativeamino acid substitutions, as defined herein. Specifically provided arechimeric antibodies containing VH and VL domains which arecamelid-derived, or human germlined variants thereof, fused to constantdomains of human antibodies, in particular human IgG1, IgG2, IgG3 orIgG4. The heavy chain variable domains defined above can be utilised assingle domain antibodies, or may be included within a conventionalfour-chain antibody or other antigen binding proteins, such as forexample Fab, Fab′, F(ab′)2, bi-specific Fab's, and Fv fragments,diabodies, linear antibodies, single-chain antibody molecules, a singlechain variable fragment (scFv) and multispecific antibodies.

Preferred embodiments of the CD70 antibodies are antibodies, or antigenbinding fragments thereof, comprising a heavy chain variable domaincomprising a variable heavy chain CDR3, a variable heavy chain CDR2 anda variable heavy chain CDR1, wherein said variable heavy chain CDR3comprises an amino acid sequence selected from the group consisting of:

SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53,SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58,SEQ ID NO:59, SEQ ID NO:262 and SEQ ID NO:263, and sequence variants ofany one of the recited sequences, wherein the sequence variant comprisesone, two or three amino acid substitutions in the recited sequence;

said variable heavy chain CDR2 optionally comprises an amino acidsequence selected from the group consisting of: amino acid sequences ofSEQ ID NO: 306 [X₁X₂X₃X₄X₅X₆X₇X₈X₉ YYADSVKX₁₀], wherein

X₁ is any amino acid, preferably D, T, S or E,

X₂ is any amino acid, preferably I,

X₃ is any amino acid, preferably N, S, T or Y,

X₄ is any amino acid, preferably N, M, S or T,

X₅ is any amino acid, preferably E, D, Y or H,

X₆ is any amino acid, preferably G, D, S or N,

X₇ is any amino acid, preferably G, Y, S, D or M,

X₈ is any amino acid, preferably T, E, S, N, Y or R,

X₉ is any amino acid, preferably T, A or R, and

X₁₀ is any amino acid, preferably G or S,

and the amino acid sequences of SEQ ID NO:30, SEQ ID NO:32, SEQ IDNO:33, SEQ ID NO:34, SEQ ID NO:249, SEQ ID NO:258 and SEQ ID NO:259 andsequence variants of any one of the recited sequences, wherein thesequence variant comprises one, two or three amino acid substitutions inthe recited sequence; and

said variable heavy chain CDR1 optionally comprises an amino acidsequence selected from the group consisting of: amino acid sequences ofSEQ ID NO: 307 [X₁ YYMN], wherein

X₁ is any amino acid, preferably V, G or A,

amino acid sequences of SEQ ID NO: 308 [X₁X₂ AMS], wherein

X₁ is any amino acid, preferably D, T, S, N or G and

X₂ is any amino acid, preferably Y, S or P,

and the amino acid sequences of SEQ ID NO:10, SEQ ID NO:15, SEQ IDNO:16, SEQ ID NO:248, SEQ ID NO:256 and SEQ ID NO:257 and sequencevariants of any one of the recited sequences, wherein the sequencevariant comprises one, two or three amino acid substitutions in therecited sequence.

The heavy chain variable domain may comprise any one of the listedvariable heavy chain CDR3 sequences (HCDR3) in combination with any oneof the variable heavy chain CDR2 sequences (HCDR2) and any one of thevariable heavy chain CDR1 sequences (HCDR1). However, certaincombinations of HCDR3 and HCDR2 and HCDR1 are particularly preferred,these being the “native” combinations which derive from a single commonVH domain. These preferred combinations are listed in Table 6 and Table14A.

The antibody or antigen binding fragment thereof may additionallycomprise a light chain variable domain (VL), which is paired with the VHdomain to form an antigen binding domain. Preferred light chain variabledomains are those comprising a variable light chain CDR3, a variablelight chain CDR2 and a variable light chain CDR1, wherein said variablelight chain CDR3 comprises an amino acid sequence selected from thegroup consisting of: SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ IDNO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO:155, SEQID NO:156, SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160,SEQ ID NO:161, SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164, SEQ IDNO:165, SEQ ID NO:166, SEQ ID NO:166, SEQ ID NO:168, SEQ ID NO:271 andSEQ ID NO:273 and sequence variants of any one of the recited sequences,wherein the sequence variant comprises one, two or three amino acidsubstitutions in the recited sequence;

said variable light chain CDR2 optionally comprises an amino acidsequence selected from the group consisting of:

(a) amino acid sequences of SEQ ID NO: 310 [X₁ TX₂X₃ RHS], wherein

X₁ is any amino acid, preferably N or S,

X₂ is any amino acid, preferably N, S or A, and

X₃ is any amino acid, preferably S, N or T,

(b) amino acid sequences of SEQ ID NO: 311 [YYSDSX₁X₂X₃ QX₄ S], wherein

X₁ is any amino acid, preferably Y, V or L,

X₂ is any amino acid, preferably K or S,

X₃ is any amino acid, preferably H or N, and

X₄ is any amino acid, preferably G or S,

(c) amino acid sequences of SEQ ID NO: 312 [X₁ NX₂ NRPS], wherein

X₁ is any amino acid, preferably V, I or Y, and

X₂ is any amino acid, preferably N or T,

(d) amino acid sequences of SEQ ID NO: 313 [GDNX₁X₂ PL], wherein

X₁ is any amino acid, preferably Y, and

X₂ is any amino acid, preferably R or M,

(e) amino acid sequences of SEQ ID NO:314 [X₁ DDX₂ RPS], wherein

X₁ is any amino acid, preferably D or G, and

X₂ is any amino acid, preferably S or I,

and the amino acid sequences of SEQ ID NO:113, SEQ ID NO:116, SEQ IDNO:120 and SEQ ID NO:270, and sequence variants of any one of therecited sequences, wherein the sequence variant comprises one, two orthree amino acid substitutions in the recited sequence; and

said variable light chain CDR1 optionally comprises an amino acidsequence selected from the group consisting of:

(a) amino acid sequences of SEQ ID NO:315 [GLX₁ SGSX₂ TX₃X₄X₅ YPX₆],wherein

X₁ is any amino acid, preferably S or T,

X₂ is any amino acid, preferably V or A,

X₃ is any amino acid, preferably S or T,

X₄ is any amino acid, preferably S, T or G,

X₅ is any amino acid, preferably N or H,

X₆ is any amino acid, preferably G, D or E,

(b) amino acid sequences of SEQ ID NO:316 [TLX₁ SX₂X₃X₄X₅ GX₆ YDIS],wherein

X₁ is any amino acid, preferably S, N or I,

X₂ is any amino acid, preferably G or A,

X₃ is any amino acid, preferably N or D,

X₄ is any amino acid, preferably N or S,

X₅ is any amino acid, preferably V or I,

X₆ is any amino acid, preferably N or S,

(c) amino acid sequences of SEQ ID NO:317 [QGGNLX₁ LYGAN], wherein

X₁ is any amino acid, preferably G or W,

(d) amino acid sequences of SEQ ID NO:318 [RGDX₁ LX₂X₃ YX₄X₅ N], wherein

X₁ is any amino acid, preferably S or T,

X₂ is any amino acid, preferably E or R,

X₃ is any amino acid, preferably R or N,

X₄ is any amino acid, preferably G or H,

X₅ is any amino acid, preferably T or A,

(e) amino acid sequences of SEQ ID NO:319 [GX₁X₂ SGSVTSX₃NFPT], wherein

X₁ is any amino acid, preferably V or L,

X₂ is any amino acid, preferably K or T,

X₃ is any amino acid, preferably T or D,

and the amino acid sequences of SEQ ID NO:82, SEQ ID NO:87, SEQ IDNO:88, SEQ ID NO:250, SEQ ID NO:251, SEQ ID NO: 252, SEQ ID NO:253, SEQID NO:267 and SEQ ID NO:268 and sequence variants of any one of therecited sequences, wherein the sequence variant comprises one, two orthree amino acid substitutions in the recited sequence.

The light chain variable domain may comprise any one of the listedvariable light chain CDR3 sequences (LCDR3) in combination with any oneof the variable light chain CDR2 sequences (LCDR2) and any one of thevariable light chain CDR1 sequences (LCDR1). However, certaincombinations of LCDR3 and LCDR2 and LCDR1 are particularly preferred,these being the “native” combinations which derive from a single commonVL domain. These preferred combinations are listed in Table 7 and Table15A.

Any given CD70 antibody or antigen binding fragment thereof comprising aVH domain paired with a VL domain to form a binding site for CD70antigen will comprise a combination of 6 CDRs: variable heavy chain CDR3(HCDR3), variable heavy chain CDR2 (HCDR2), variable heavy chain CDR1(HCDR1), variable light chain CDR3 (LCDR3), variable light chain CDR2(LCDR2) and variable light chain CDR1 (LCDR1). Although all combinationsof 6 CDRs selected from the CDR sequence groups listed above arepermissible, and within the scope of the invention, certain combinationsof 6 CDRs are particularly preferred; these being the “native”combinations within a single Fab exhibiting high affinity binding tohuman CD70.

Preferred combinations of 6 CDRs include, but are not limited to, thecombinations of variable heavy chain CDR3 (HCDR3), variable heavy chainCDR2 (HCDR2), variable heavy chain CDR1 (HCDR1), variable light chainCDR3 (LCDR3), variable light chain CDR2 (LCDR2) and variable light chainCDR1 (LCDR1) selected from the group consisting of:

(i) HCDR3 comprising SEQ ID NO:50, HCDR2 comprising SEQ ID NO:27, HCDR1comprising SEQ ID NO:11, LCDR3 comprising SEQ ID NO:160, LCDR2comprising SEQ ID NO:119, and LCDR1 comprising SEQ ID NO:250;

(ii) HCDR3 comprising SEQ ID NO:49, HCDR2 comprising SEQ ID NO:26, HCDR1comprising SEQ ID NO:10, LCDR3 comprising SEQ ID NO:148, LCDR2comprising SEQ ID NO:109, and LCDR1 comprising SEQ ID NO:77;

(iii) HCDR3 comprising SEQ ID NO:50, HCDR2 comprising SEQ ID NO:27,HCDR1 comprising SEQ ID NO:11, LCDR3 comprising SEQ ID NO:149, LCDR2comprising SEQ ID NO:110, and LCDR1 comprising SEQ ID NO:78;

(iv) HCDR3 comprising SEQ ID NO:50, HCDR2 comprising SEQ ID NO:28, HCDR1comprising SEQ ID NO:11, LCDR3 comprising SEQ ID NO:150, LCDR2comprising SEQ ID NO:111, and LCDR1 comprising SEQ ID NO:79;

(v) HCDR3 comprising SEQ ID NO:50, HCDR2 comprising SEQ ID NO:28, HCDR1comprising SEQ ID NO:11, LCDR3 comprising SEQ ID NO:151, LCDR2comprising SEQ ID NO:110, and LCDR1 comprising SEQ ID NO:80;

(vi) HCDR3 comprising SEQ ID NO:51, HCDR2 comprising SEQ ID NO:29, HCDR1comprising SEQ ID NO:12, LCDR3 comprising SEQ ID NO: 152, LCDR2comprising SEQ ID NO:110, and LCDR1 comprising SEQ ID NO:80;

(vii) HCDR3 comprising SEQ ID NO:52, HCDR2 comprising SEQ ID NO:30,HCDR1 comprising SEQ ID NO:13, LCDR3 comprising SEQ ID NO:153, LCDR2comprising SEQ ID NO: 112, and LCDR1 comprising SEQ ID NO:81;

(viii) HCDR3 comprising SEQ ID NO:53, HCDR2 comprising SEQ ID NO:31,HCDR1 comprising SEQ ID NO:14, LCDR3 comprising SEQ ID NO:154, LCDR2comprising SEQ ID NO:113, and LCDR1 comprising SEQ ID NO:82;

(ix) HCDR3 comprising SEQ ID NO:54, HCDR2 comprising SEQ ID NO:32, HCDR1comprising SEQ ID NO:15, LCDR3 comprising SEQ ID NO:155, LCDR2comprising SEQ ID NO:114, and LCDR1 comprising SEQ ID NO:83;

(x) HCDR3 comprising SEQ ID NO:55, HCDR2 comprising SEQ ID NO:33, HCDR1comprising SEQ ID NO:16, LCDR3 comprising SEQ ID NO:156, LCDR2comprising SEQ ID NO:115, and LCDR1 comprising SEQ ID NO:84;

(xi) HCDR3 comprising SEQ ID NO:56, HCDR2 comprising SEQ ID NO:34, HCDR1comprising SEQ ID NO:17, LCDR3 comprising SEQ ID NO:157, LCDR2comprising SEQ ID NO:116, and LCDR1 comprising SEQ ID NO:85;

(xii) HCDR3 comprising SEQ ID NO:57, HCDR2 comprising SEQ ID NO:35,HCDR1 comprising SEQ ID NO: 18, LCDR3 comprising SEQ ID NO:158, LCDR2comprising SEQ ID NO: 117, and LCDR1 comprising SEQ ID NO: 84;

(xiii) HCDR3 comprising SEQ ID NO: 58, HCDR2 comprising SEQ ID NO: 36,HCDR1 comprising SEQ ID NO: 19, LCDR3 comprising SEQ ID NO: 159, LCDR2comprising SEQ ID NO: 118, and LCDR1 comprising SEQ ID NO: 86;

(xiv) HCDR3 comprising SEQ ID NO: 50, HCDR2 comprising SEQ ID NO: 27,HCDR1 comprising SEQ ID NO: 11, LCDR3 comprising SEQ ID NO: 161, LCDR2comprising SEQ ID NO:120, and LCDR1 comprising SEQ ID NO:88;

(xv) HCDR3 comprising SEQ ID NO: 50, HCDR2 comprising SEQ ID NO: 27,HCDR1 comprising SEQ ID NO: 11, LCDR3 comprising SEQ ID NO: 162, LCDR2comprising SEQ ID NO: 121, and LCDR1 comprising SEQ ID NO: 89;

(xvi) HCDR3 comprising SEQ ID NO: 50, HCDR2 comprising SEQ ID NO: 27,HCDR1 comprising SEQ ID NO: 11, LCDR3 comprising SEQ ID NO: 163, LCDR2comprising SEQ ID NO: 122, and LCDR1 comprising SEQ ID NO: 90;

(xvii) HCDR3 comprising SEQ ID NO: 51, HCDR2 comprising SEQ ID NO: 29,HCDR1 comprising SEQ ID NO: 12, LCDR3 comprising SEQ ID NO: 164, LCDR2comprising SEQ ID NO: 123, and LCDR1 comprising SEQ ID NO:91;

(xviii) HCDR3 comprising SEQ ID NO: 51, HCDR2 comprising SEQ ID NO: 29,HCDR1 comprising SEQ ID NO: 12, LCDR3 comprising SEQ ID NO: 164, LCDR2comprising SEQ ID NO: 124, and LCDR1 comprising SEQ ID NO: 91;

(xix) HCDR3 comprising SEQ ID NO: 59, HCDR2 comprising SEQ ID NO: 37,HCDR1 comprising SEQ ID NO: 12, LCDR3 comprising SEQ ID NO: 165, LCDR2comprising SEQ ID NO: 125, and LCDR1 comprising SEQ ID NO: 92;

(xx) HCDR3 comprising SEQ ID NO: 59, HCDR2 comprising SEQ ID NO: 37,HCDR1 comprising SEQ ID NO: 20, LCDR3 comprising SEQ ID NO: 165, LCDR2comprising SEQ ID NO: 126, and LCDR1 comprising SEQ ID NO: 93;

(xxi) HCDR3 comprising SEQ ID NO: 59, HCDR2 comprising SEQ ID NO: 37,HCDR1 comprising SEQ ID NO: 20, LCDR3 comprising SEQ ID NO: 166, LCDR2comprising SEQ ID NO: 127, and LCDR1 comprising SEQ ID NO: 92;

(xxii) HCDR3 comprising SEQ ID NO:59, HCDR2 comprising SEQ ID NO: 37,HCDR1 comprising SEQ ID NO: 20, LCDR3 comprising SEQ ID NO: 167, LCDR2comprising SEQ ID NO: 128, and LCDR1 comprising SEQ ID NO: 94;

(xxiii) HCDR3 comprising SEQ ID NO: 59, HCDR2 comprising SEQ ID NO: 37,HCDR1 comprising SEQ ID NO: 20, LCDR3 comprising SEQ ID NO: 168, LCDR2comprising SEQ ID NO: 110, and LCDR1 comprising SEQ ID NO: 95;

(xxiv) HCDR3 comprising SEQ ID NO: 262, HCDR2 comprising SEQ ID NO: 258,HCDR1 comprising SEQ ID NO: 256, LCDR3 comprising SEQ ID NO: 271, LCDR2comprising SEQ ID NO: 110, and LCDR1 comprising SEQ ID NO: 267;

(xxv) HCDR3 comprising SEQ ID NO: 263, HCDR2 comprising SEQ ID NO: 259,HCDR1 comprising SEQ ID NO: 257, LCDR3 comprising SEQ ID NO: 273, LCDR2comprising SEQ ID NO: 270, and LCDR1 comprising SEQ ID NO: 268.

Further preferred CD70 antibodies, exhibiting high affinity binding tohuman CD70, include isolated antibodies or antigen binding fragmentsthereof, comprising a heavy chain variable domain having an amino acidsequence selected from the group consisting of: the amino acid sequencesof SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:179, SEQ ID NO:180, SEQ IDNO:181, SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:184, SEQ ID NO:185, SEQID NO:186, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:212, SEQ ID NO:213,SEQ ID NO:214, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ IDNO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQID NO:223, SEQ ID NO:224, SEQ ID NO:225, SEQ ID NO:226, SEQ ID NO:227,SEQ ID NO:228, SEQ ID NO:229, SEQ ID NO:274 and SEQ ID NO:275 and aminoacid sequences exhibiting at least 90%, 95%, 97%, 98% or 99% sequenceidentity to one of the recited sequences, and optionally comprising alight chain variable domain having an amino acid sequence selected fromthe group consisting of: the amino acid sequences of SEQ ID NO:189, SEQID NO:190, SEQ ID NO:191, SEQ ID NO:192, SEQ ID NO:193, SEQ ID NO:194,SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:198, SEQ IDNO:199, SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, SEQID NO:204, SEQ ID NO:205, SEQ ID NO:206, SEQ ID NO:207, SEQ ID NO:208,SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211, SEQ ID NO:223, SEQ IDNO:230, SEQ ID NO:231, SEQ ID NO:232, SEQ ID NO:233, SEQ ID NO:234, SEQID NO:234, SEQ ID NO:236, SEQ ID NO:237, SEQ ID NO:238, SEQ ID NO:239,SEQ ID NO:240, SEQ ID NO:241, SEQ ID NO:242, SEQ ID NO:243, SEQ IDNO:244, SEQ ID NO:245, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:248, SEQID NO:276 and SEQ ID NO:277 and amino acid sequences exhibiting at least90%, 95%, 97%, 98% or 99% sequence identity to one of the recitedsequences.

Although all possible pairings of VH domains and VL domains selectedfrom the VH and VL domain sequence groups listed above are permissible,and within the scope of the invention, certain combinations VH and VLare particularly preferred; these being the “native” combinations withina single Fab exhibiting high affinity binding to human CD70.Accordingly, preferred CD70 antibodies, or antigen binding fragmentsthereof, exhibiting high affinity CD70 binding are those comprising acombination of a heavy chain variable domain (VH) and a light chainvariable domain (VL), wherein the combination is selected from the groupconsisting of:

(i) a heavy chain variable domain comprising the amino acid sequence ofSEQ ID NO:223 and a light chain variable domain comprising the aminoacid sequence of SEQ ID NO:241;

(ii) a heavy chain variable domain comprising the amino acid sequence ofSEQ ID NO:177 and a light chain variable domain comprising the aminoacid sequence of SEQ ID NO:189;

(iii) a heavy chain variable domain comprising the amino acid sequenceof SEQ ID NO:178 and a light chain variable domain comprising the aminoacid sequence of SEQ ID NO: 190;

(iv) a heavy chain variable domain comprising the amino acid sequence ofSEQ ID NO:179 and a light chain variable domain comprising the aminoacid sequence of SEQ ID NO:191;

(v) a heavy chain variable domain comprising the amino acid sequence ofSEQ ID NO:179 and a light chain variable domain comprising the aminoacid sequence of SEQ ID NO:192;

(vi) a heavy chain variable domain comprising the amino acid sequence ofSEQ ID NO:180 and a light chain variable domain comprising the aminoacid sequence of SEQ ID NO:193;

(vii) a heavy chain variable domain comprising the amino acid sequenceof SEQ ID NO:181 and a light chain variable domain comprising the aminoacid sequence of SEQ ID NO:194;

(viii) a heavy chain variable domain comprising the amino acid sequenceof SEQ ID NO:182 and a light chain variable domain comprising the aminoacid sequence of SEQ ID NO:195;

(ix) a heavy chain variable domain comprising the amino acid sequence ofSEQ ID NO:183 and a light chain variable domain comprising the aminoacid sequence of SEQ ID NO:196;

(x) a heavy chain variable domain comprising the amino acid sequence ofSEQ ID NO:184 and a light chain variable domain comprising the aminoacid sequence of SEQ ID NO:197;

(xi) a heavy chain variable domain comprising the amino acid sequence ofSEQ ID NO:185 and a light chain variable domain comprising the aminoacid sequence of SEQ ID NO:198;

(xii) a heavy chain variable domain comprising the amino acid sequenceof SEQ ID NO:186 and a light chain variable domain comprising the aminoacid sequence of SEQ ID NO:199;

(xiii) a heavy chain variable domain comprising the amino acid sequenceof SEQ ID NO:187 and a light chain variable domain comprising the aminoacid sequence of SEQ ID NO:200;

(xiv) a heavy chain variable domain comprising the amino acid sequenceof SEQ ID NO:178 and a light chain variable domain comprising the aminoacid sequence of SEQ ID NO:201;

(xv) a heavy chain variable domain comprising the amino acid sequence ofSEQ ID NO:178 and a light chain variable domain comprising the aminoacid sequence of SEQ ID NO:202;

(xvi) a heavy chain variable domain comprising the amino acid sequenceof SEQ ID NO:178 and a light chain variable domain comprising the aminoacid sequence of SEQ ID NO:203;

(xvii) a heavy chain variable domain comprising the amino acid sequenceof SEQ ID NO:178 and a light chain variable domain comprising the aminoacid sequence of SEQ ID NO:204;

(xviii) a heavy chain variable domain comprising the amino acid sequenceof SEQ ID NO:180 and a light chain variable domain comprising the aminoacid sequence of SEQ ID NO:205;

(xix) a heavy chain variable domain comprising the amino acid sequenceof SEQ ID NO:180 and a light chain variable domain comprising the aminoacid sequence of SEQ ID NO:206;

(xx) a heavy chain variable domain comprising the amino acid sequence ofSEQ ID NO:188 and a light chain variable domain comprising the aminoacid sequence of SEQ ID NO:207;

(xxi) a heavy chain variable domain comprising the amino acid sequenceof SEQ ID NO:188 and a light chain variable domain comprising the aminoacid sequence of SEQ ID NO:208;

(xxii) a heavy chain variable domain comprising the amino acid sequenceof SEQ ID NO:188 and a light chain variable domain comprising the aminoacid sequence of SEQ ID NO:209;

(xxiii) a heavy chain variable domain comprising the amino acid sequenceof SEQ ID NO:188 and a light chain variable domain comprising the aminoacid sequence of SEQ ID NO:210;

(xxiv) a heavy chain variable domain comprising the amino acid sequenceof SEQ ID NO:188 and a light chain variable domain comprising the aminoacid sequence of SEQ ID NO:211;

(xxv) a heavy chain variable domain comprising the amino acid sequenceof SEQ ID NO:274 and a light chain variable domain comprising the aminoacid sequence of SEQ ID NO:276;

(xxvi) a heavy chain variable domain comprising the amino acid sequenceof SEQ ID NO:275 and a light chain variable domain comprising the aminoacid sequence of SEQ ID NO:277.

For each of the specific VH/VL combinations listed above, it is alsopermissible, and within the scope of the invention, to combine a VHdomain having an amino acid sequence at least 90%, 92%, 95%, 97% or 99%identical to the recited VH domain sequence with a VL domain having anamino acid sequence at least 90%, 92%, 95%, 97% or 99% identical to therecited VL domain sequence.

In the preceding paragraph, and elsewhere herein, the structure of theantibodies/antigen binding fragments is defined on the basis of %sequence identity with a recited reference sequence (with a given SEQ IDNO). In this context, % sequence identity between two amino acidsequences may be determined by comparing these two sequences aligned inan optimum manner and in which the amino acid sequence to be comparedcan comprise additions or deletions with respect to the referencesequence for an optimum alignment between these two sequences. Thepercentage of identity is calculated by determining the number ofidentical positions for which the amino acid residue is identicalbetween the two sequences, by dividing this number of identicalpositions by the total number of positions in the comparison window andby multiplying the result obtained by 100 in order to obtain thepercentage of identity between these two sequences. Typically, thecomparison window with correspond to the full length of the sequencebeing compared. For example, it is possible to use the BLAST program,“BLAST 2 sequences” (Tatusova et al, “Blast 2 sequences—a new tool forcomparing protein and nucleotide sequences”, FEMS Microbiol Lett.174:247-250) available on the sitehttp://www.ncbi.nlm.nih.gov/gorf/b12.html, the parameters used beingthose given by default (in particular for the parameters “open gappenalty”: 5, and “extension gap penalty”: 2; the matrix chosen being,for example, the matrix “BLOSUM 62” proposed by the program), thepercentage of identity between the two sequences to be compared beingcalculated directly by the program.

The most preferred CD70 antibodies provided herein, which exhibit aparticularly advantageous combination of properties, including extremelyhigh affinity binding to human CD70, are those based on theantigen-binding portion of the llama-derived Fab denoted 27B3 in theaccompanying examples, plus human germlined variants of 27B3, includingthe germlined variants identified in the accompanying examples. 27B3 andits germlined variants, particularly variants based on the CDRs orcomplete variable domains of variant 41D12, exhibit an extremelyadvantageous combination of properties, summarised as follows: highaffinity binding to recombinant human CD70, strong binding to cellsurface CD70, specifically binding to CD70 expressed on cancer celllines, particularly cell lines which express CD70 at “low copy number”and strong binding to cancer cells isolated from patient samples (CLL),potent blocking of the CD70/CD27 interaction, potent effectorfunction—particularly when expressed as a chimera with human IgG1constant regions, and especially when expressed as a non-fucosylatedIgG1, cross-reactivity with CD70 homologs of rhesus macaque andcynomolgus monkey enabling toxicology studies in primate species,binding to both native (i.e. cell-surface) and heat denatured CD70,partial or low levels of internalisation on certain cancer cell lines.All of these characteristics in combination render 41D12, and indeedother 27B3 variants and the other CD70 antibodies described herein whichexhibit similar properties, an outstanding candidate for therapeutic usein the treatment of CD70-associated diseases, specificallyCD70-expressing cancers and immunological disorders.

27B3, and its variants, are characterised by the presence of the heavychain variable CDR3 sequence shown as SEQ ID NO:50 (DAGYSNHVPIFDS).

Accordingly, preferred embodiments of the CD70 antibody, or antigenbinding fragments thereof, are those comprising a heavy chain variabledomain wherein the variable heavy chain CDR3 comprises or consists ofthe amino acid sequence of SEQ ID NO:50 or a sequence variant thereof,wherein the sequence variant comprises one, two or three amino acidsubstitutions in the recited sequence.

More preferred embodiments are antibodies or antigen binding fragmentsthereof which include the same combination of heavy chain CDRs as 27B3,or human germlined variants of 27B3. Accordingly, the antibody orantigen binding fragment thereof, may comprise a heavy chain variabledomain wherein the variable heavy chain CDR3 comprises or consists ofthe amino acid sequence of SEQ ID NO:50 or a sequence variant thereof;

the variable heavy chain CDR2 comprises or consists of an amino acidsequence selected from the group consisting of: SEQ ID NO:27, SEQ IDNO:249 and sequence variants thereof; and

the variable heavy chain CDR1 comprises or consists of an amino acidsequence selected from the group consisting of: SEQ ID NO:11, SEQ IDNO:248 and sequence variants thereof, wherein the sequence variantscomprise one, two or three amino acid substitutions (e.g., conservativesubstitutions, humanising substitutions or affinity variants) in therecited sequence.

Any combination of HCDR3, HCDR2 and HCDR1 from within the recited groupsof CDR sequences is permissible, and within the scope of the invention,however certain combinations are particular preferred. Accordingly, inpreferred embodiments the antigen or antigen binding fragment thereofmay comprise a heavy chain variable domain wherein the combination ofHCDR3, HCDR2 and HCDR1 is selected from the following:

-   -   (a) the variable heavy chain CDR3 comprises or consists of SEQ        ID NO:50 (DAGYSNHVPIFDS) or a sequence variant thereof;        -   the variable heavy chain CDR2 comprises or consists of SEQ            ID NO:27 (DINNEGGTTYYADSVKG) or a sequence variant thereof;            and        -   the variable heavy chain CDR1 comprises or consists of SEQ            ID NO:11 (VYYMN) or a sequence variant thereof,        -   wherein the sequence variants comprises one, two or three            amino acid substitutions (e.g., conservative substitutions,            humanising substitutions or affinity variants) in the            recited sequence;    -   (b) the variable heavy chain CDR3 comprises or consists of SEQ        ID NO:50 (RDAGYSNHVPIFDS) or a sequence variant thereof;        -   the variable heavy chain CDR2 comprises or consists of SEQ            ID NO:249 (DINNEGGATYYADSVKG) or a sequence variant thereof;            and        -   the variable heavy chain CDR1 comprises or consists of SEQ            ID NO:11 (VYYMN) or a sequence variant thereof,        -   wherein the sequence variants comprises one, two or three            amino acid substitutions (e.g., conservative substitutions,            humanising substitutions or affinity variants) in the            recited sequence.    -   (c) the variable heavy chain CDR3 comprises or consists of SEQ        ID NO:50 (DAGYSNHVPIFDS) or a sequence variant thereof;        -   the variable heavy chain CDR2 comprises or consists of SEQ            ID NO:27 (DINNEGGTTYYADSVKG) or a sequence variant thereof;            and        -   the variable heavy chain CDR1 comprises or consists of SEQ            ID NO:248 (GYYMN) or a sequence variant thereof,        -   wherein the sequence variants comprises one, two or three            amino acid substitutions (e.g., conservative substitutions,            humanising substitutions or affinity variants) in the            recited sequence.

In preferred embodiments, the antibody or antigen binding fragmentthereof also includes a light chain variable domain (VL), paired withthe heavy chain variable domain, In the preferred light chain variabledomains, the variable light chain CDR3 comprises or consists of SEQ IDNO:160 or a sequence variant thereof;

the variable light chain CDR2 comprises or consists of an amino acidsequence selected from the group consisting of: SEQ ID NO:119, SEQ IDNO:110 and sequence variants of the recited sequences; and

the variable light chain CDR1 comprises or consists of an amino acidsequence selected from the group consisting of: SEQ ID NO:87, SEQ IDNO:250, SEQ ID NO:251, SEQ ID NO:252, SEQ ID NO:253 and sequencevariants of the recited sequences, and

wherein the sequence variants comprise one, two or three amino acidsubstitutions (e.g., conservative substitutions, humanisingsubstitutions or affinity variants) in the recited sequences.

Any combination of LCDR3, LCDR2 and LCDR1 from within the recited groupsof light chain CDR sequences is permissible, and within the scope of theinvention, however certain combinations are particular preferred.Accordingly, in preferred embodiments the antigen or antigen bindingfragment thereof may comprise a light chain variable domain wherein thecombination of LCDR3, LCDR2 and LCDR1 is selected from the following:

-   -   (a) the variable light chain CDR3 comprises or consists of SEQ        ID NO:160 (ALFISNPSVE) or a sequence variant thereof;        -   the variable light chain CDR2 comprises or consists of SEQ            ID NO:119 (NTNTRHS) or a sequence variant thereof; and        -   the variable light chain CDR1 comprises or consists of SEQ            ID NO:250 (GLKSGSVTSDNFPT) or a sequence variant thereof,        -   wherein the sequence variants comprises one, two or three            amino acid substitutions (e.g., conservative substitutions,            humanising substitutions or affinity variants) in the            recited sequence;    -   (b) the variable light chain CDR3 comprises or consists of SEQ        ID NO:160 (ALFISNPSVE) or a sequence variant thereof;        -   the variable light chain CDR2 comprises or consists of SEQ            ID NO:119 (NTNTRHS) or a sequence variant thereof; and        -   the variable light chain CDR1 comprises or consists of SEQ            ID NO:87 (GLKSGSVTSTNFPT) or a sequence variant thereof,        -   wherein the sequence variants comprises one, two or three            amino acid substitutions (e.g., conservative substitutions,            humanising substitutions or affinity variants) in the            recited sequence.

Other preferred light chain CDR combinations for human “germlined”variants of 27B3 are given in Table 15A.

The most preferred embodiment of the CD70 antibody or antigen bindingfragment thereof comprises a heavy chain variable domain (VH) and alight chain variable domain (VL) wherein the combination of 6 CDRs whichforms the binding site for human CD70 is as follows:

the variable heavy chain CDR3 comprises or consists of SEQ ID NO:50(DAGYSNHVPIFDS) or a sequence variant thereof;

the variable heavy chain CDR2 comprises or consists of SEQ ID NO:27(DINNEGGTTYYADSVKG) or a sequence variant thereof;

the variable heavy chain CDR1 comprises or consists of SEQ ID NO:11(VYYMN) or a sequence variant thereof;

the variable light chain CDR3 comprises or consists of SEQ ID NO:160(ALFISNPSVE) or a sequence variant thereof;

the variable light chain CDR2 comprises or consists of SEQ ID NO:119(NTNTRHS) or a sequence variant thereof; and

the variable light chain CDR1 comprises or consists of SEQ ID NO:250(GLKSGSVTSDNFPT) or a sequence variant thereof,

wherein the sequence variants comprises one, two or three amino acidsubstitutions (e.g., conservative substitutions, humanisingsubstitutions or affinity variants) in the recited sequence.

Other preferred combinations of 6 CDRs are those “native” combinationswhich occur in the human germlined variants of 27B3 listed in Tables 14A(heavy chains) and 15A (light chains).

In the foregoing preferred embodiments based on 27B3 and its germlinedvariants, the antibody preferably includes the CH1 domain, hinge region,CH2 domain and CH3 domain of a human antibody, in particular human IgG1,IgG2, IgG3 or IgG4. The most preferred embodiment is a human IgG1. It isstill further preferred for the human IgG1 to be engineering to maximiseeffector function in one or more of antibody-dependent cellularcytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) orantibody-dependent cellular phagocytosis (ADCP). Particularly preferredis a non-fucosylated human IgG1, e.g. a non-fucosylated IgG1 producedusing the Potelligent™ technology of BioWa Inc.

Further preferred CD70 antibodies, exhibiting high affinity binding tohuman CD70, based on the Fab denoted 27B3 and human germlined variantsof 27B3, include isolated antibodies or antigen binding fragmentsthereof, comprising a heavy chain variable domain having an amino acidsequence selected from the group consisting of: the amino acid sequencesof SEQ ID NO:178, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, SEQ IDNO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219, SEQID NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:223, SEQ ID NO:224,SEQ ID NO:225, SEQ ID NO:226, SEQ ID NO:227, SEQ ID NO:228, SEQ IDNO:229, SEQ ID NO:274 and SEQ ID NO:275 and amino acid sequencesexhibiting at least 90%, 95%, 97%, 98% or 99% sequence identity to oneof the recited sequences, and optionally comprising a light chainvariable domain having an amino acid sequence selected from the groupconsisting of: the amino acid sequences of SEQ ID NO:201, SEQ ID NO:230,SEQ ID NO:231, SEQ ID NO:232, SEQ ID NO:233, SEQ ID NO:234, SEQ IDNO:234, SEQ ID NO:236, SEQ ID NO:237, SEQ ID NO:238, SEQ ID NO:239, SEQID NO:240, SEQ ID NO:241, SEQ ID NO:242, SEQ ID NO:243, SEQ ID NO:244,SEQ ID NO:245, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:248, SEQ IDNO:276 and SEQ ID NO:277 and amino acid sequences exhibiting at least90%, 95%, 97%, 98% or 99% sequence identity to one of the recitedsequences.

Although all possible pairings of VH domains and VL domains selectedfrom the VH and VL domain sequence groups listed above are permissible,and within the scope of the invention, certain combinations VH and VLare particularly preferred; these being the “native” combinations withina single Fab exhibiting high affinity binding to human CD70. In the caseof the germlined variants of 27B3 recited in Tables 14B and 15B, it maybe preferred to retain the original VH/VL pairing. Accordingly,preferred CD70 antibodies, or antigen binding fragments thereof,exhibiting high affinity CD70 binding are those comprising a combinationof a heavy chain variable domain and a light chain variable domain,wherein the combination is selected from the group consisting of:

(i) a heavy chain variable domain comprising the amino acid sequence ofSEQ ID NO:223 and a light chain variable domain comprising the aminoacid sequence of SEQ ID NO:241;

(ii) a heavy chain variable domain comprising the amino acid sequence ofSEQ ID NO:178 and a light chain variable domain comprising the aminoacid sequence of SEQ ID NO: 190;

(iii) a heavy chain variable domain comprising the amino acid sequenceof SEQ ID NO:212 and a light chain variable domain comprising the aminoacid sequence of SEQ ID NO: 230

(iv) a heavy chain variable domain comprising the amino acid sequence ofSEQ ID NO:213 and a light chain variable domain comprising the aminoacid sequence of SEQ ID NO:231;

(v) a heavy chain variable domain comprising the amino acid sequence ofSEQ ID NO:214 and a light chain variable domain comprising the aminoacid sequence of SEQ ID NO:232;

(vi) a heavy chain variable domain comprising the amino acid sequence ofSEQ ID NO:215 and a light chain variable domain comprising the aminoacid sequence of SEQ ID NO:235;

(vii) a heavy chain variable domain comprising the amino acid sequenceof SEQ ID NO:216 and a light chain variable domain comprising the aminoacid sequence of SEQ ID NO:234;

(viii) a heavy chain variable domain comprising the amino acid sequenceof SEQ ID NO:217 and a light chain variable domain comprising the aminoacid sequence of SEQ ID NO:235;

(ix) a heavy chain variable domain comprising the amino acid sequence ofSEQ ID NO:218 and a light chain variable domain comprising the aminoacid sequence of SEQ ID NO:236;

(x) a heavy chain variable domain comprising the amino acid sequence ofSEQ ID NO:219 and a light chain variable domain comprising the aminoacid sequence of SEQ ID NO:237;

(xi) a heavy chain variable domain comprising the amino acid sequence ofSEQ ID NO:220 and a light chain variable domain comprising the aminoacid sequence of SEQ ID NO:238;

(xii) a heavy chain variable domain comprising the amino acid sequenceof SEQ ID NO:221 and a light chain variable domain comprising the aminoacid sequence of SEQ ID NO:239;

(xiii) a heavy chain variable domain comprising the amino acid sequenceof SEQ ID NO:222 and a light chain variable domain comprising the aminoacid sequence of SEQ ID NO:240;

(xiv) a heavy chain variable domain comprising the amino acid sequenceof SEQ ID NO:224 and a light chain variable domain comprising the aminoacid sequence of SEQ ID NO:242;

(xv) a heavy chain variable domain comprising the amino acid sequence ofSEQ ID NO:225 and a light chain variable domain comprising the aminoacid sequence of SEQ ID NO:243;

(xvi) a heavy chain variable domain comprising the amino acid sequenceof SEQ ID NO:226 and a light chain variable domain comprising the aminoacid sequence of SEQ ID NO:244;

(xvii) a heavy chain variable domain comprising the amino acid sequenceof SEQ ID NO:227 and a light chain variable domain comprising the aminoacid sequence of SEQ ID NO:245;

(xviii) a heavy chain variable domain comprising the amino acid sequenceof SEQ ID NO:228 and a light chain variable domain comprising the aminoacid sequence of SEQ ID NO:246;

(xix) a heavy chain variable domain comprising the amino acid sequenceof SEQ ID NO:229 and a light chain variable domain comprising the aminoacid sequence of SEQ ID NO:247;

(xx) a heavy chain variable domain comprising the amino acid sequence ofSEQ ID NO:223 and a light chain variable domain comprising the aminoacid sequence of SEQ ID NO:244;

(xxi) a heavy chain variable domain comprising the amino acid sequenceof SEQ ID NO:223 and a light chain variable domain comprising the aminoacid sequence of SEQ ID NO:245.

For each of the specific VH/VL combinations listed above, it is alsopermissible, and within the scope of the invention, to combine a VHdomain having an amino acid sequence at least 90%, 92%, 95%, 97% or 99%identical to the recited VH domain sequence with a VL domain having anamino acid sequence at least 90%, 92%, 95%, 97% or 99% identical to therecited VL domain sequence.

The most preferred embodiment is a CD70 antibody or antigen bindingfragment thereof based on the native VH/VL combination of the humangermlined variant denoted 41D12. Accordingly, there is also providedherein an antibody or antigen binding fragment thereof comprising aheavy chain variable domain (VH) comprising or consisting of an aminoacid sequence selected from the group consisting of: the amino acidsequence shown as SEQ ID NO:223, germlined variants and affinityvariants thereof and amino acid sequences at least 90%, 95%, 97%, 98% or99% identical thereto, and a light chain variable domain (VL) comprisingor consisting of an amino acid sequence selected from the groupconsisting of: the amino acid sequence shown as SEQ ID NO:241, germlinedvariants and affinity variants thereof and amino acid sequences at least90%, 95%, 97%, 98% or 99% identical thereto.

Embodiments wherein the amino acid sequence of the VH domain exhibitsless than 100% sequence identity with the sequence shown as SEQ ID NO:223 may nevertheless comprise heavy chain CDRs which are identical toHCDR1, HCDR2 and HCDR3 of SEQ ID NO:223 (SEQ ID NOs:11, 27 and 50,respectively) whilst exhibiting amino acid sequence variation within theframework regions. Likewise, embodiments wherein the amino acid sequenceof the VL domain exhibits less than 100% sequence identity with thesequence shown as SEQ ID NO: 241 may nevertheless comprise heavy chainCDRs which are identical to LCDR1, LCDR2 and LCDR3 of SEQ ID NO:241 (SEQID NOs:250, 116 and 160, respectively) whilst exhibiting amino acidsequence variation within the framework regions.

In the foregoing preferred embodiments based on the VH and VL domains of41D12, or variants thereof, the antibody preferably includes the CH1domain, hinge region, CH2 domain and CH3 domain of a human antibody, inparticular human IgG1, IgG2, IgG3 or IgG4. The most preferred embodimentis a human IgG1. It is still further preferred for the human IgG1 to beengineering to maximise effector function in one or more of ADCC, CDC orADCP. Particularly preferred is a de-fucosylated human IgG1, preferablyprepared using the Potelligent™ expression system.

Another advantageous embodiment is an antibody or antigen bindingfragment thereof comprising a heavy chain variable domain (VH)comprising or consisting of an amino acid sequence selected from thegroup consisting of: the amino acid sequence shown as SEQ ID NO:225,germlined variants and affinity variants thereof and amino acidsequences at least 90%, 95%, 97%, 98% or 99% identical thereto, and alight chain variable domain (VL) comprising or consisting of an aminoacid sequence selected from the group consisting of: the amino acidsequence shown as SEQ ID NO:243, germlined variants and affinityvariants thereof and amino acid sequences at least 90%, 95%, 97%, 98% or99% identical thereto; or

Another advantageous embodiment is an antibody or antigen bindingfragment thereof comprising a heavy chain variable domain (VH)comprising or consisting of an amino acid sequence selected from thegroup consisting of: the amino acid sequence shown as SEQ ID NO:226,germlined variants and affinity variants thereof and amino acidsequences at least 90%, 95%, 97%, 98% or 99% identical thereto, and alight chain variable domain (VL) comprising or consisting of an aminoacid sequence selected from the group consisting of: the amino acidsequence shown as SEQ ID NO:244, germlined variants and affinityvariants thereof and amino acid sequences at least 90%, 95%, 97%, 98% or99% identical thereto.

Features/Properties of CD70 Antibodies

In the aforementioned aspects and embodiments, the CD70 antibodies, orantigen binding fragments thereof, may each exhibit one or more, or anycombination, of the following properties or features:

The antibody or antigen binding fragment may bind to human CD70 withhigh affinity, exhibiting an off-rate for human CD70 of 7×10⁻⁴ s⁻¹ orless, preferably 5×10⁻⁴ s⁻¹ or less, and typically in the range of from0.4×10⁻⁴ s⁻¹ to 4.8×10⁻⁴ s⁻¹, when tested as a Fab fragment.

The antibody or antigen binding fragment may bind to human CD70 withhigh affinity and inhibit the interaction between CD70 and CD27.Alternatively, the antibody or antigen binding fragment may bind tohuman CD70 but not inhibit the interaction between CD70 and CD27.

The antibody or antigen binding fragment may bind with high affinity tohuman CD70 on the surface of CD70-expressing cells.

The antibody or antigen binding fragment may bind to human CD70 on thesurface of CD70-expressing cells and be slowly or only partiallyinternalised. A key aspect of the invention is the observation that theCD70 antibodies are in fact very poorly internalised on a large numberof CD70-expressing cell-lines, including many CD70-expressing cancercell lines. This observation is in direct contrast to previous publishedreports that CD70 antibodies are rapidly internalised following bindingto renal cell carcinoma cell lines (see Adam et al., British Journal ofCancer (2006) 95: 298-306; and WO 2007/038637) and has directimplications for therapeutic use of the antibodies. The observation thatthe CD70 antibodies are very poorly internalised following binding tocancer cells strongly supports the conclusion that therapeuticstrategies for treatment of many CD70-expressing cancers, and indeedCD70-associated immunological diseases, should be based on extreme highaffinity binding to CD70, coupled with antibody effector function, inparticular any one or more of ADCC, CDC or ADCP, and not on the use ofimmunoconjugates in which the CD70 antibody is linked to a therapeuticagent, e.g. a cytotoxic or cytostatic drug moiety.

The antibody or antigen binding fragment may bind within the amino acidsequence HIQVTLAICSS (SEQ ID NO:342) in human CD70;

The antibody or antigen binding fragment may exhibit cross-reactivitywith CD70 of simian origin, specifically the CD70 homologs of rhesusmacaque (Macaca mulatta) and cynomolgus monkey (Macaca cynomolgus).

The antibody or antigen binding fragment may bind to both native humanCD70 (e.g. CD70 expressed on the surface of a cell, such as a cell lineor a CD70-expressing cell isolated from a human patient) and heatdenatured recombinant human CD70.

The antibody or antigen binding fragment may provide very highproduction yields (>4 g/L) in recombinant antibody expression systems,such as for example the CHK1SV cell line (proprietary to BioWa/Lonza),as compared to a 1-2 g/L historical average for therapeutic antibodyproducts, resulting in a substantial reduction in production costs.

The antibody or antigen binding fragment may be highly stable under 37°C. storage conditions and in freeze-thaw cycles, which is also a majorcost reduction factor.

The antibody may exhibit one or more effector functions selected fromantibody-dependent cell-mediated cytotoxicity (ADCC), complementdependent cytotoxicity (CDC) and antibody-dependent cell-mediatedphagocytosis (ADCP) against cells expressing human CD70 protein on thecell surface.

The antibody may exhibit ADCC against CD70-expressing cells, e.g. cancercells or other malignant cells, or immune cells.

The antibody may exhibit enhanced ADCC function in comparison to areference antibody which is an equivalent antibody comprising a nativehuman Fc domain. In a non-limiting embodiment, the ADCC function may beat least 10× enhanced in comparison to the reference antibody comprisinga native human Fc domain. In this context “equivalent” may be taken tomean that the antibody with enhanced ADCC function displayssubstantially identical antigen-binding specificity and/or sharesidentical amino acid sequence with the reference antibody, except forany modifications made (relative to native human Fc) for the purposes ofenhancing ADCC.

The antibody or antigen binding fragment may inhibit tumour growth in anin vivo tumour xenograft model, in the absence of conjugation to acytotoxic or cytostatic agent. In a non-limiting embodiment, theinhibition of tumour growth function may be at least 10 fold enhanced incomparison to the reference antibody SGN70.

The antibody or antigen binding fragment may induce apoptosis ofCD70-expressing cells.

The antibody may contain the hinge region, CH2 domain and CH3 domain ofa human IgG, most preferably human IgG1, IgG2, IgG3 or IgG4.

The antibody may include modifications in the Fc region, such asmodifications which enhance antibody effector function as explainedelsewhere herein. In particular, the antibody may be a non-fucosylatedIgG.

In further aspects, the invention also provides polynucleotide moleculeswhich encode the above-listed CD70 antibodies and antigen bindingfragments thereof, in addition to expression vectors comprising thepolynucleotides, host cells containing the vectors and methods ofrecombinant expression/production of the CD70 antibodies.

In a still further aspect, the invention provides a pharmaceuticalcomposition comprising any one of the CD70 antibodies described aboveand a pharmaceutically acceptable carrier or excipient.

A still further aspect of the invention concerns methods of medicaltreatment using the above-listed CD70 antibodies, particularly in thetreatment of cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be further understood with reference to the followingexperimental examples and the accompanying Figures in which:

FIG. 1: Shows the immune response tested in ELISA on recombinant CD70for llamas immunised with 786-O cells (top) and with Raji cells(bottom).

FIG. 2: illustrates inhibition of binding of CD27 to CD70 byllama-derived CD70 Fabs and reference CD70 Fabs, measured by ELISA.

FIG. 3: is a graphical representation of the signal for llama-derivedFabs tested in a binding Elisa (black) or in an inhibition Elisa(white).

FIG. 4: illustrates inhibition of binding of CD27 to human CD70 (A&B) orrhesus CD70 (C) by chimeric llama-human CD70 mAbs and reference CD70mAbs measured by ELISA.

FIG. 5: shows binding of chimeric llama-human CD70 mAbs to 786-O cells(A) or MHH-PREB-1 cells (B) as demonstrated by FACS analysis.

FIG. 6: shows inhibition by CD70 specific chimeric llama-human mAbs in aRaji cell based co-culture potency assay.

FIG. 7: shows the results of standard Cr⁵¹ release ADCC assay on 786-Ocells.

FIG. 8: shows the results of CDC assay on U266 cells in the presence of9% human serum.

FIG. 9: demonstrates the efficacy of chimeric llama-human CD70 mAbs in aADCP assay on 786-O cells

FIG. 10. illustrates antibody internalisation, assessed as MFI OUT fordifferent chimeric llama-human CD70 mAbs as a function of time on 786-Ocells in two independent experiments.

FIG. 11: demonstrates survival of mice in a Raji Xenograft model aftertreatment with chimeric llama-human CD70 mAb 41D12, isotype control andFc-dead control.

FIG. 12: depicts alignments of the VH and VL amino acid sequences ofclone 27B3 with the amino acid sequences of human germline gene segmentsVH3-38 and VL8-61, respectively.

FIG. 13: shows gelfiltration analysis of samples of germlined 27B3 mAbvariants taken after 5 weeks incubation at 37° C.

FIG. 14: shows the potency in CD70 binding, as measured using Biacore,of samples of germlined CD70 mAbs taken at various time points afterincubation at various temperatures.

FIG. 15: shows the binding affinity of CD70 mAbs for CD70-expressingcancer cell lines.

FIG. 16: shows the affinity of CD70 mAbs for CLL patient cellsexpressing CD70.

FIG. 17: shows lysis of SU-DHL-6 cells bound by CD70 mAbs.

FIG. 18: shows inhibition of binding of CD27 to CD70 by CD70 mAbs,measured by ELISA.

FIG. 19: shows an alignment of CD70 sequences from different species.

FIG. 20: shows the binding affinity of CD70 mAbs for human U266 cells,rhesus monkey LCL8864 cells and cynomologus monkey HSC-F cells.

FIG. 21: shows inhibition of binding of CD27 to CD70 of human, rhesusmonkey and cynomologus monkey by CD70 mAbs as determined by ELISA.

FIG. 22: shows binding of CD70 mAbs to denatured recombinant CD70,assessed by ELISA.

FIG. 23: shows CD70 chimeric sequences used for epitope mapping.

FIG. 24: illustrates antibody internalisation for CD70 mAbs as afunction of time on 786-O cells.

DEFINITIONS

“Antibody” or “Immunoglobulin”—As used herein, the term “immunoglobulin”includes a polypeptide having a combination of two heavy and two lightchains whether or not it possesses any relevant specificimmunoreactivity. “Antibodies” refers to such assemblies which havesignificant known specific immunoreactive activity to an antigen ofinterest (e.g. human CD70). The term “CD70 antibodies” is used herein torefer to antibodies which exhibit immunological specificity for humanCD70 protein. As explained elsewhere herein, “specificity” for humanCD70 does not exclude cross-reaction with species homologues of CD70.Antibodies and immunoglobulins comprise light and heavy chains, with orwithout an interchain covalent linkage between them. Basicimmunoglobulin structures in vertebrate systems are relatively wellunderstood.

The generic term “immunoglobulin” comprises five distinct classes ofantibody that can be distinguished biochemically. All five classes ofantibodies are within the scope of the present invention, the followingdiscussion will generally be directed to the IgG class of immunoglobulinmolecules. With regard to IgG, immunoglobulins comprise two identicallight polypeptide chains of molecular weight approximately 23,000Daltons, and two identical heavy chains of molecular weight53,000-70,000. The four chains are joined by disulfide bonds in a “Y”configuration wherein the light chains bracket the heavy chains startingat the mouth of the “Y” and continuing through the variable region.

The light chains of an antibody are classified as either kappa or lambda(κ, λ). Each heavy chain class may be bound with either a kappa orlambda light chain. In general, the light and heavy chains arecovalently bonded to each other, and the “tail” portions of the twoheavy chains are bonded to each other by covalent disulfide linkages ornon-covalent linkages when the immunoglobulins are generated either byhybridomas, B cells or genetically engineered host cells. In the heavychain, the amino acid sequences run from an N-terminus at the forkedends of the Y configuration to the C-terminus at the bottom of eachchain. Those skilled in the art will appreciate that heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, (γ, μ, α, δ, ε) withsome subclasses among them (e.g., γ1-γ4). It is the nature of this chainthat determines the “class” of the antibody as IgG, IgM, IgA, IgD orIgE, respectively. The immunoglobulin subclasses (isotypes) e.g., IgG1,IgG2, IgG3, IgG4, IgA1, etc. are well characterized and are known toconfer functional specialization. Modified versions of each of theseclasses and isotypes are readily discernable to the skilled artisan inview of the instant disclosure and, accordingly, are within the scope ofthe instant invention.

As indicated above, the variable region of an antibody allows theantibody to selectively recognize and specifically bind epitopes onantigens. That is, the VL domain and VH domain of an antibody combine toform the variable region that defines a three dimensional antigenbinding site. This quaternary antibody structure forms the antigenbinding site present at the end of each arm of the Y. More specifically,the antigen binding site is defined by three complementary determiningregions (CDRs) on each of the VH and VL chains.

“CD70 protein” or “CD70 antigen”—As used herein, the terms “CD70protein” or “CD70 antigen” or “CD70” are used interchangeably and referto a member of the TNF ligand family which is a ligand forTNFRSF27/CD27. The terms “human CD70 protein” or “human CD70 antigen” or“human CD70” are used interchangeably to refer specifically to the humanhomolog, including the native human CD70 protein naturally expressed inthe human body and/or on the surface of cultured human cell lines, aswell as recombinant forms and fragments thereof. Specific examples ofhuman CD70 include the polypeptide having the amino acid sequence shownunder NCBI Reference Sequence Accession No. NP_001243, or theextracellular domain thereof.“Binding Site”—As used herein, the term “binding site” comprises aregion of a polypeptide which is responsible for selectively binding toa target antigen of interest (e.g. human CD70). Binding domains compriseat least one binding site. Exemplary binding domains include an antibodyvariable domain. The antibody molecules of the invention may comprise asingle binding site or multiple (e.g., two, three or four) bindingsites.“Derived From”—As used herein the term “derived from” a designatedprotein (e.g. a CD70 antibody or antigen-binding fragment thereof)refers to the origin of the polypeptide. In one embodiment, thepolypeptide or amino acid sequence which is derived from a particularstarting polypeptide is a CDR sequence or sequence related thereto. Inone embodiment, the amino acid sequence which is derived from aparticular starting polypeptide is not contiguous. For example, in oneembodiment, one, two, three, four, five, or six CDRs are derived from astarting antibody. In one embodiment, the polypeptide or amino acidsequence which is derived from a particular starting polypeptide oramino acid sequence has an amino acid sequence that is essentiallyidentical to that of the starting sequence, or a portion thereof whereinthe portion consists of at least of at least 3-5 amino acids, 5-10 aminoacids, at least 10-20 amino acids, at least 20-30 amino acids, or atleast 30-50 amino acids, or which is otherwise identifiable to one ofordinary skill in the art as having its origin in the starting sequence.In one embodiment, the one or more CDR sequences derived from thestarting antibody are altered to produce variant CDR sequences, e.g.affinity variants, wherein the variant CDR sequences maintain CD70binding activity.“Camelid-Derived”—In certain preferred embodiments, the CD70 antibodymolecules of the invention comprise framework amino acid sequencesand/or CDR amino acid sequences derived from a camelid conventionalantibody raised by active immunisation of a camelid with CD70 antigen.However, CD70 antibodies comprising camelid-derived amino acid sequencesmay be engineered to comprise framework and/or constant region sequencesderived from a human amino acid sequence (i.e. a human antibody) orother non-camelid mammalian species. For example, a human or non-humanprimate framework region, heavy chain portion, and/or hinge portion maybe included in the subject CD70 antibodies. In one embodiment, one ormore non-camelid amino acids may be present in the framework region of a“camelid-derived” CD70 antibody, e.g., a camelid framework amino acidsequence may comprise one or more amino acid mutations in which thecorresponding human or non-human primate amino acid residue is present.Moreover, camelid-derived VH and VL domains, or humanised variantsthereof, may be linked to the constant domains of human antibodies toproduce a chimeric molecule, as extensively described elsewhere herein.“Conservative amino acid substitution”—A “conservative amino acidsubstitution” is one in which the amino acid residue is replaced with anamino acid residue having a similar side chain. Families of amino acidresidues having similar side chains have been defined in the art,including basic side chains (e.g., lysine, arginine, histidine), acidicside chains (e.g., aspartic acid, glutamic acid), uncharged polar sidechains (e.g., glycine, asparagine, glutamine, serine, threonine,tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Thus, a nonessential amino acid residue in an immunoglobulinpolypeptide may be replaced with another amino acid residue from thesame side chain family. In another embodiment, a string of amino acidscan be replaced with a structurally similar string that differs in orderand/or composition of side chain family members.“Heavy chain portion”—As used herein, the term “heavy chain portion”includes amino acid sequences derived from the constant domains of animmunoglobulin heavy chain. A polypeptide comprising a heavy chainportion comprises at least one of: a CH1 domain, a hinge (e.g., upper,middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain,or a variant or fragment thereof. In one embodiment, an antibody orantigen binding fragment of the invention may comprise the Fc portion ofan immunoglobulin heavy chain (e.g., a hinge portion, a CH2 domain, anda CH3 domain). In another embodiment, an antibody or antigen bindingfragment of the invention may lack at least a portion of a constantdomain (e.g., all or part of a CH2 domain). In certain embodiments, atleast one, and preferably all, of the constant domains are derived froma human immunoglobulin heavy chain. For example, in one preferredembodiment, the heavy chain portion comprises a fully human hingedomain. In other preferred embodiments, the heavy chain portioncomprising a fully human Fc portion (e.g., hinge, CH2 and CH3 domainsequences from a human immunoglobulin).

In certain embodiments, the constituent constant domains of the heavychain portion are from different immunoglobulin molecules. For example,a heavy chain portion of a polypeptide may comprise a CH2 domain derivedfrom an IgG1 molecule and a hinge region derived from an IgG3 or IgG4molecule. In other embodiments, the constant domains are chimericdomains comprising portions of different immunoglobulin molecules. Forexample, a hinge may comprise a first portion from an IgG1 molecule anda second portion from an IgG3 or IgG4 molecule. As set forth above, itwill be understood by one of ordinary skill in the art that the constantdomains of the heavy chain portion may be modified such that they varyin amino acid sequence from the naturally occurring (wild-type)immunoglobulin molecule. That is, the polypeptides of the inventiondisclosed herein may comprise alterations or modifications to one ormore of the heavy chain constant domains (CH1, hinge, CH2 or CH3) and/orto the light chain constant region domain (CL). Exemplary modificationsinclude additions, deletions or substitutions of one or more amino acidsin one or more domains.

“Chimeric”—A “chimeric” protein comprises a first amino acid sequencelinked to a second amino acid sequence with which it is not naturallylinked in nature. The amino acid sequences may normally exist inseparate proteins that are brought together in the fusion polypeptide orthey may normally exist in the same protein but are placed in a newarrangement in the fusion polypeptide. A chimeric protein may becreated, for example, by chemical synthesis, or by creating andtranslating a polynucleotide in which the peptide regions are encoded inthe desired relationship. Exemplary chimeric CD70 antibodies includefusion proteins comprising camelid-derived VH and VL domains, orhumanised variants thereof, fused to the constant domains of a humanantibody, e.g. human IgG1, IgG2, IgG3 or IgG4.“Variable region” or “variable domain”—The terms “variable region” and“variable domain” are used herein interchangeable and are intended tohave equivalent meaning. The term “variable” refers to the fact thatcertain portions of the variable domains VH and VL differ extensively insequence among antibodies and are used in the binding and specificity ofeach particular antibody for its target antigen. However, thevariability is not evenly distributed throughout the variable domains ofantibodies. It is concentrated in three segments called “hypervariableloops” in each of the VL domain and the VH domain which form part of theantigen binding site. The first, second and third hypervariable loops ofthe VLambda light chain domain are referred to herein as L1(λ), L2(λ)and L3(λ) and may be defined as comprising residues 24-33 (L1(λ),consisting of 9, 10 or 11 amino acid residues), 49-53 (L2(λ), consistingof 3 residues) and 90-96 (L3(λ), consisting of 5 residues) in the VLdomain (Morea et al., Methods 20:267-279 (2000)). The first, second andthird hypervariable loops of the VKappa light chain domain are referredto herein as L1(κ), L2(κ) and L3(κ) and may be defined as comprisingresidues 25-33 (L1(κ), consisting of 6, 7, 8, 11, 12 or 13 residues),49-53 (L2(κ), consisting of 3 residues) and 90-97 (L3(κ), consisting of6 residues) in the VL domain (Morea et al., Methods 20:267-279 (2000)).The first, second and third hypervariable loops of the VH domain arereferred to herein as H1, H2 and H3 and may be defined as comprisingresidues 25-33 (H1, consisting of 7, 8 or 9 residues), 52-56 (H2,consisting of 3 or 4 residues) and 91-105 (H3, highly variable inlength) in the VH domain (Morea et al., Methods 20:267-279 (2000)).

Unless otherwise indicated, the terms L1, L2 and L3 respectively referto the first, second and third hypervariable loops of a VL domain, andencompass hypervariable loops obtained from both Vkappa and Vlambdaisotypes. The terms H1, H2 and H3 respectively refer to the first,second and third hypervariable loops of the VH domain, and encompasshypervariable loops obtained from any of the known heavy chain isotypes,including γ, ε, δ, α or μ.

The hypervariable loops L1, L2, L3, H1, H2 and H3 may each comprise partof a “complementarity determining region” or “CDR”, as defined below.The terms “hypervariable loop” and “complementarity determining region”are not strictly synonymous, since the hypervariable loops (HVs) aredefined on the basis of structure, whereas complementarity determiningregions (CDRs) are defined based on sequence variability (Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md., 1983) and thelimits of the HVs and the CDRs may be different in some VH and VLdomains.

The CDRs of the VL and VH domains can typically be defined as comprisingthe following amino acids: residues 24-34 (CDRL1), 50-56 (CDRL2) and89-97 (CDRL3) in the light chain variable domain, and residues 31-35 or31-35b (CDRH1), 50-65 (CDRH2) and 95-102 (CDRH3) in the heavy chainvariable domain; (Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md. (1991)). Thus, the HVs may be comprised within thecorresponding CDRs and references herein to the “hypervariable loops” ofVH and VL domains should be interpreted as also encompassing thecorresponding CDRs, and vice versa, unless otherwise indicated.

The more highly conserved portions of variable domains are called theframework region (FR), as defined below. The variable domains of nativeheavy and light chains each comprise four FRs (FR1, FR2, FR3 and FR4,respectively), largely adopting a (3-sheet configuration, connected bythe three hypervariable loops. The hypervariable loops in each chain areheld together in close proximity by the FRs and, with the hypervariableloops from the other chain, contribute to the formation of theantigen-binding site of antibodies. Structural analysis of antibodiesrevealed the relationship between the sequence and the shape of thebinding site formed by the complementarity determining regions (Chothiaet al., J. Mol. Biol. 227: 799-817 (1992)); Tramontano et al., J. Mol.Biol, 215:175-182 (1990)). Despite their high sequence variability, fiveof the six loops adopt just a small repertoire of main-chainconformations, called “canonical structures”. These conformations arefirst of all determined by the length of the loops and secondly by thepresence of key residues at certain positions in the loops and in theframework regions that determine the conformation through their packing,hydrogen bonding or the ability to assume unusual main-chainconformations.

“CDR”—As used herein, the term “CDR” or “complementarity determiningregion” means the non-contiguous antigen combining sites found withinthe variable region of both heavy and light chain polypeptides. Theseparticular regions have been described by Kabat et al., J. Biol. Chem.252, 6609-6616 (1977) and Kabat et al., Sequences of protein ofimmunological interest. (1991), and by Chothia et al., J. Mol. Biol.196:901-917 (1987) and by MacCallum et al., J. Mol. Biol. 262:732-745(1996) where the definitions include overlapping or subsets of aminoacid residues when compared against each other. The amino acid residueswhich encompass the CDRs as defined by each of the above citedreferences are set forth for comparison. Preferably, the term “CDR” is aCDR as defined by Kabat based on sequence comparisons.

TABLE 1 CDR definitions CDR Definitions Kabat¹ Chothia² MacCallum³ V_(H)CDR1 31-35 26-32 30-35 V_(H) CDR2 50-65 53-55 47-58 V_(H) CDR3  95-102 96-101  93-101 V_(L) CDR1 24-34 26-32 30-36 V_(L) CDR2 50-56 50-5246-55 V_(L) CDR3 89-97 91-96 89-96 ¹Residue numbering follows thenomenclature of Kabat et al., supra ²Residue numbering follows thenomenclature of Chothia et al., supra ³Residue numbering follows thenomenclature of MacCallum et al., supra“Framework region”—The term “framework region” or “FR region” as usedherein, includes the amino acid residues that are part of the variableregion, but are not part of the CDRs (e.g., using the Kabat definitionof CDRs). Therefore, a variable region framework is between about100-120 amino acids in length but includes only those amino acidsoutside of the CDRs. For the specific example of a heavy chain variabledomain and for the CDRs as defined by Kabat et al., framework region 1corresponds to the domain of the variable region encompassing aminoacids 1-30; framework region 2 corresponds to the domain of the variableregion encompassing amino acids 36-49; framework region 3 corresponds tothe domain of the variable region encompassing amino acids 66-94, andframework region 4 corresponds to the domain of the variable region fromamino acids 103 to the end of the variable region. The framework regionsfor the light chain are similarly separated by each of the light claimvariable region CDRs. Similarly, using the definition of CDRs by Chothiaet al. or McCallum et al. the framework region boundaries are separatedby the respective CDR termini as described above. In preferredembodiments the CDRs are as defined by Kabat.

In naturally occurring antibodies, the six CDRs present on eachmonomeric antibody are short, non-contiguous sequences of amino acidsthat are specifically positioned to form the antigen binding site as theantibody assumes its three dimensional configuration in an aqueousenvironment. The remainder of the heavy and light variable domains showless inter-molecular variability in amino acid sequence and are termedthe framework regions. The framework regions largely adopt a β-sheetconformation and the CDRs form loops which connect, and in some casesform part of, the β-sheet structure. Thus, these framework regions actto form a scaffold that provides for positioning the six CDRs in correctorientation by inter-chain, non-covalent interactions. The antigenbinding site formed by the positioned CDRs defines a surfacecomplementary to the epitope on the immunoreactive antigen. Thiscomplementary surface promotes the non-covalent binding of the antibodyto the immunoreactive antigen epitope. The position of CDRs can bereadily identified by one of ordinary skill in the art.

“Hinge region”—As used herein, the term “hinge region” includes theportion of a heavy chain molecule that joins the CH1 domain to the CH2domain. This hinge region comprises approximately 25 residues and isflexible, thus allowing the two N-terminal antigen binding regions tomove independently. Hinge regions can be subdivided into three distinctdomains: upper, middle, and lower hinge domains (Roux et al. J. Immunol.1998 161:4083). CD70 antibodies comprising a “fully human” hinge regionmay contain one of the hinge region sequences shown in Table 2 below.

TABLE 2 human hinge sequences IgG Upper hinge Middle hinge Lower hingeIgG1 EPKSCDKTHT CPPCP APELLGGP (SEQ ID NO: 320) (SEQ ID NO: 321)(SEQ ID NO: 322) IgG3 ELKTPLGDTTHT CPRCP (EPKSCDTPPPCPRCP)₃ APELLGGP(SEQ ID NO: 323) (SEQ ID NO: 324) (SEQ ID NO: 325) IgG4 ESKYGPP CPSCPAPEFLGGP (SEQ ID NO: 326) (SEQ ID NO: 327) (SEQ ID NO: 328) IgG42 ERKCCVECPPPCP APPVAGP (SEQ ID NO: 329) (SEQ ID NO: 330) (SEQ ID NO: 331)“CH2 domain”—As used herein the term “CH2 domain” includes the portionof a heavy chain molecule that extends, e.g., from about residue 244 toresidue 360 of an antibody using conventional numbering schemes(residues 244 to 360, Kabat numbering system; and residues 231-340, EUnumbering system, Kabat E A et al. Sequences of Proteins ofImmunological Interest. Bethesda, US Department of Health and HumanServices, NIH. 1991). The CH2 domain is unique in that it is not closelypaired with another domain. Rather, two N-linked branched carbohydratechains are interposed between the two CH2 domains of an intact nativeIgG molecule. It is also well documented that the CH3 domain extendsfrom the CH2 domain to the C-terminal of the IgG molecule and comprisesapproximately 108 residues.“Fragment”—The term “fragment” refers to a part or portion of anantibody or antibody chain comprising fewer amino acid residues than anintact or complete antibody or antibody chain. The term “antigen-bindingfragment” refers to a polypeptide fragment of an immunoglobulin orantibody that binds antigen or competes with intact antibody (i.e., withthe intact antibody from which they were derived) for antigen binding(i.e., specific binding to human CD70). As used herein, the term“fragment” of an antibody molecule includes antigen-binding fragments ofantibodies, for example, an antibody light chain variable domain (VL),an antibody heavy chain variable domain (VH), a single chain antibody(scFv), a F(ab′)2 fragment, a Fab fragment, an Fd fragment, an Fvfragment, and a single domain antibody fragment (DAb). Fragments can beobtained, e.g., via chemical or enzymatic treatment of an intact orcomplete antibody or antibody chain or by recombinant means.“Valency”—As used herein the term “valency” refers to the number ofpotential target binding sites in a polypeptide. Each target bindingsite specifically binds one target molecule or specific site on a targetmolecule. When a polypeptide comprises more than one target bindingsite, each target binding site may specifically bind the same ordifferent molecules (e.g., may bind to different ligands or differentantigens, or different epitopes on the same antigen). The subjectbinding molecules have at least one binding site specific for a humanCD70 molecule.“Specificity”—The term “specificity” refers to the ability to bind(e.g., immunoreact with) a given target, e.g., CD70. A polypeptide maybe monospecific and contain one or more binding sites which specificallybind a target or a polypeptide may be multispecific and contain two ormore binding sites which specifically bind the same or differenttargets. In one embodiment, an antibody of the invention is specific formore than one target. For example, in one embodiment, a multispecificbinding molecule of the invention binds to CD70 and a second moleculeexpressed on a tumor cell. Exemplary antibodies which comprise antigenbinding sites that bind to antigens expressed on tumor cells are knownin the art and one or more CDRs from such antibodies can be included inan antibody of the invention.“Synthetic”—As used herein the term “synthetic” with respect topolypeptides includes polypeptides which comprise an amino acid sequencethat is not naturally occurring. For example, non-naturally occurringpolypeptides which are modified forms of naturally occurringpolypeptides (e.g., comprising a mutation such as an addition,substitution or deletion) or which comprise a first amino acid sequence(which may or may not be naturally occurring) that is linked in a linearsequence of amino acids to a second amino acid sequence (which may ormay not be naturally occurring) to which it is not naturally linked innature.“Engineered”—As used herein the term “engineered” includes manipulationof nucleic acid or polypeptide molecules by synthetic means (e.g. byrecombinant techniques, in vitro peptide synthesis, by enzymatic orchemical coupling of peptides or some combination of these techniques).Preferably, the antibodies of the invention are engineered, includingfor example, humanized and/or chimeric antibodies, and antibodies whichhave been engineered to improve one or more properties, such as antigenbinding, stability/half-life or effector function.“Modified antibody”—As used herein, the term “modified antibody”includes synthetic forms of antibodies which are altered such that theyare not naturally occurring, e.g., antibodies that comprise at least twoheavy chain portions but not two complete heavy chains (such as, domaindeleted antibodies or minibodies); multispecific forms of antibodies(e.g., bispecific, trispecific, etc.) altered to bind to two or moredifferent antigens or to different epitopes on a single antigen); heavychain molecules joined to scFv molecules and the like. ScFv moleculesare known in the art and are described, e.g., in U.S. Pat. No.5,892,019. In addition, the term “modified antibody” includesmultivalent forms of antibodies (e.g., trivalent, tetravalent, etc.,antibodies that bind to three or more copies of the same antigen). Inanother embodiment, a modified antibody of the invention is a fusionprotein comprising at least one heavy chain portion lacking a CH2 domainand comprising a binding domain of a polypeptide comprising the bindingportion of one member of a receptor ligand pair.

The term “modified antibody” may also be used herein to refer to aminoacid sequence variants of a CD70 antibody. It will be understood by oneof ordinary skill in the art that a CD70 antibody may be modified toproduce a variant CD70 antibody which varies in amino acid sequence incomparison to the CD70 antibody from which it was derived. For example,nucleotide or amino acid substitutions leading to conservativesubstitutions or changes at “non-essential” amino acid residues may bemade (e.g., in CDR and/or framework residues). Amino acid substitutionscan include replacement of one or more amino acids with a naturallyoccurring or non-natural amino acid.

“Humanising substitutions”—As used herein, the term “humanisingsubstitutions” refers to amino acid substitutions in which the aminoacid residue present at a particular position in the VH or VL domain ofa CD70 antibody (for example a camelid-derived CD70 antibody) isreplaced with an amino acid residue which occurs at an equivalentposition in a reference human VH or VL domain. The reference human VH orVL domain may be a VH or VL domain encoded by the human germline.Humanising substitutions may be made in the framework regions and/or theCDRs of a CD70 antibody, defined herein.“Humanised variants”—As used herein the term “humanised variant” refersto a variant antibody which contains one or more “humanisingsubstitutions” compared to a reference CD70 antibody, wherein a portionof the reference antibody (e.g. the VH domain and/or the VL domain orparts thereof containing at least one CDR) has an amino derived from anon-human species, and the “humanising substitutions” occur within theamino acid sequence derived from a non-human species.“Germlined variants”—The term “germlined variant” is used herein torefer specifically to “humanised variants” in which the “humanisingsubstitutions” result in replacement of one or more amino acid residuespresent at a particular position (s) in the VH or VL domain of a CD70antibody (for example a camelid-derived CD70 antibody) with an aminoacid residue which occurs at an equivalent position in a reference humanVH or VL domain encoded by the human germline. It is typical that forany given “germlined variant”, the replacement amino acid residuessubstituted into the germlined variant are taken exclusively, orpredominantly, from a single human germline-encoded VH or VL domain. Theterms “humanised variant” and “germlined variant” are often usedinterchangeably herein. Introduction of one or more “humanisingsubstitutions” into a camelid-derived (e.g. llama derived) VH or VLdomain results in production of a “humanised variant” of the camelid(llama)-derived VH or VL domain. If the amino acid residues substitutedin are derived predominantly or exclusively from a single humangermline-encoded VH or VL domain sequence, then the result may be a“human germlined variant” of the camelid (llama)-derived VH or VLdomain.“Affinity variants”—As used herein, the term “affinity variant” refersto a variant antibody which exhibits one or more changes in amino acidsequence compared to a reference CD70 antibody, wherein the affinityvariant exhibits an altered affinity for the human CD70 protein incomparison to the reference antibody. Typically, affinity variants willexhibit a changed affinity for human CD70, as compared to the referenceCD70 antibody. Preferably the affinity variant will exhibit improvedaffinity for human CD70, as compared to the reference CD70 antibody. Theimprovement may be apparent as a lower K_(D), for human CD70, or aslower off-rate for human CD70 or an alteration in the pattern ofcross-reactivity with non-human CD70 homologues. Affinity variantstypically exhibit one or more changes in amino acid sequence in theCDRs, as compared to the reference CD70 antibody. Such substitutions mayresult in replacement of the original amino acid present at a givenposition in the CDRs with a different amino acid residue, which may be anaturally occurring amino acid residue or a non-naturally occurringamino acid residue. The amino acid substitutions may be conservative ornon-conservative.“High human homology”—An antibody comprising a heavy chain variabledomain (VH) and a light chain variable domain (VL) will be considered ashaving high human homology if the VH domains and the VL domains, takentogether, exhibit at least 90% amino acid sequence identity to theclosest matching human germline VH and VL sequences. Antibodies havinghigh human homology may include antibodies comprising VH and VL domainsof native non-human antibodies which exhibit sufficiently high %sequence identity to human germline sequences, including for exampleantibodies comprising VH and VL domains of camelid conventionalantibodies, as well as engineered, especially humanised or germlined,variants of such antibodies and also “fully human” antibodies.

In one embodiment the VH domain of the antibody with high human homologymay exhibit an amino acid sequence identity or sequence homology of 80%or greater with one or more human VH domains across the frameworkregions FR1, FR2, FR3 and FR4. In other embodiments the amino acidsequence identity or sequence homology between the VH domain of thepolypeptide of the invention and the closest matching human germline VHdomain sequence may be 85% or greater, 90% or greater, 95% or greater,97% or greater, or up to 99% or even 100%.

In one embodiment the VH domain of the antibody with high human homologymay contain one or more (e.g. 1 to 10) amino acid sequence mis-matchesacross the framework regions FR1, FR2, FR3 and FR4, in comparison to theclosest matched human VH sequence.

In another embodiment the VL domain of the antibody with high humanhomology may exhibit a sequence identity or sequence homology of 80% orgreater with one or more human VL domains across the framework regionsFR1, FR2, FR3 and FR4. In other embodiments the amino acid sequenceidentity or sequence homology between the VL domain of the polypeptideof the invention and the closest matching human germline VL domainsequence may be 85% or greater 90% or greater, 95% or greater, 97% orgreater, or up to 99% or even 100%.

In one embodiment the VL domain of the antibody with high human homologymay contain one or more (e.g. 1 to 10) amino acid sequence mis-matchesacross the framework regions FR1, FR2, FR3 and FR4, in comparison to theclosest matched human VL sequence.

Before analyzing the percentage sequence identity between the antibodywith high human homology and human germline VH and VL, the canonicalfolds may be determined, which allows the identification of the familyof human germline segments with the identical combination of canonicalfolds for H1 and H2 or L1 and L2 (and L3). Subsequently the humangermline family member that has the highest degree of sequence homologywith the variable region of the antibody of interest is chosen forscoring the sequence homology. The determination of Chothia canonicalclasses of hypervariable loops L1, L2, L3, H1 and H2 can be performedwith the bioinformatics tools publicly available on webpagewww.bioinf.org.uk/abs/chothia.html.page. The output of the program showsthe key residue requirements in a datafile. In these datafiles, the keyresidue positions are shown with the allowed amino acids at eachposition. The sequence of the variable region of the antibody ofinterest is given as input and is first aligned with a consensusantibody sequence to assign the Kabat numbering scheme. The analysis ofthe canonical folds uses a set of key residue templates derived by anautomated method developed by Martin and Thornton (Martin et al., J.Mol. Biol. 263:800-815 (1996)).

With the particular human germline V segment known, which uses the samecombination of canonical folds for H1 and H2 or L1 and L2 (and L3), thebest matching family member in terms of sequence homology can bedetermined. With bioinformatics tools the percentage sequence identitybetween the VH and VL domain framework amino acid sequences of theantibody of interest and corresponding sequences encoded by the humangermline can be determined, but actually manual alignment of thesequences can be applied as well. Human immunoglobulin sequences can beidentified from several protein data bases, such as VBase(http://vbase.mrc-cpe.cam.ac.uk/) or the Pluckthun/Honegger database(http://www.bioc.unizh.ch/antibody/Sequences/Germlines. To compare thehuman sequences to the V regions of VH or VL domains in an antibody ofinterest a sequence alignment algorithm such as available via websiteslike www.expasy.ch/tools/#align can be used, but also manual alignmentwith the limited set of sequences can be performed. Human germline lightand heavy chain sequences of the families with the same combinations ofcanonical folds and with the highest degree of homology with theframework regions 1, 2, and 3 of each chain are selected and comparedwith the variable region of interest; also the FR4 is checked againstthe human germline JH and JK or JL regions.

Note that in the calculation of overall percent sequence homology theresidues of FR1, FR2 and FR3 are evaluated using the closest matchsequence from the human germline family with the identical combinationof canonical folds. Only residues different from the closest match orother members of the same family with the same combination of canonicalfolds are scored (NB—excluding any primer-encoded differences). However,for the purposes of humanization, residues in framework regionsidentical to members of other human germline families, which do not havethe same combination of canonical folds, can be considered “human”,despite the fact that these are scored “negative” according to thestringent conditions described above. This assumption is based on the“mix and match” approach for humanization, in which each of FR1, FR2,FR3 and FR4 is separately compared to its closest matching humangermline sequence and the humanized molecule therefore contains acombination of different FRs as was done by Qu and colleagues (Qu etla., Clin. Cancer Res. 5:3095-3100 (1999)) and Ono and colleagues (Onoet al., Mol. Immunol. 36:387-395 (1999)). The boundaries of theindividual framework regions may be assigned using the IMGT numberingscheme, which is an adaptation of the numbering scheme of Chothia(Lefranc et al., NAR 27: 209-212 (1999); imgt.cines.fr).

Antibodies with high human homology may comprise hypervariable loops orCDRs having human or human-like canonical folds, as discussed in detailbelow. In one embodiment at least one hypervariable loop or CDR ineither the VH domain or the VL domain of the antibody with high humanhomology may be obtained or derived from a VH or VL domain of anon-human antibody, for example a conventional antibody from a speciesof Camelidae, yet exhibit a predicted or actual canonical fold structurewhich is substantially identical to a canonical fold structure whichoccurs in human antibodies.

It is well established in the art that although the primary amino acidsequences of hypervariable loops present in both VH domains and VLdomains encoded by the human germline are, by definition, highlyvariable, all hypervariable loops, except CDR H3 of the VH domain, adoptonly a few distinct structural conformations, termed canonical folds(Chothia et al., J. Mol. Biol. 196:901-917 (1987); Tramontano et al.Proteins 6:382-94 (1989)), which depend on both the length of thehypervariable loop and presence of the so-called canonical amino acidresidues (Chothia et al., J. Mol. Biol. 196:901-917 (1987)). Actualcanonical structures of the hypervariable loops in intact VH or VLdomains can be determined by structural analysis (e.g. X-raycrystallography), but it is also possible to predict canonical structureon the basis of key amino acid residues which are characteristic of aparticular structure (discussed further below). In essence, the specificpattern of residues that determines each canonical structure forms a“signature” which enables the canonical structure to be recognised inhypervariable loops of a VH or VL domain of unknown structure; canonicalstructures can therefore be predicted on the basis of primary amino acidsequence alone.

The predicted canonical fold structures for the hypervariable loops ofany given VH or VL sequence in an antibody with high human homology canbe analysed using algorithms which are publicly available fromwww.bioinf.org.uk/abs/chothia.html,www.biochem.ucl.ac.uk/˜martin/antibodies.html andwww.bioc.unizh.ch/antibody/Sequences/Germlines/Vbase_hVk.html. Thesetools permit query VH or VL sequences to be aligned against human VH orVL domain sequences of known canonical structure, and a prediction ofcanonical structure made for the hypervariable loops of the querysequence.

In the case of the VH domain, H1 and H2 loops may be scored as having acanonical fold structure “substantially identical” to a canonical foldstructure known to occur in human antibodies if at least the first, andpreferable both, of the following criteria are fulfilled:

1. An identical length, determined by the number of residues, to theclosest matching human canonical structural class.

2. At least 33% identity, preferably at least 50% identity with the keyamino acid residues described for the corresponding human H1 and H2canonical structural classes.

(note for the purposes of the foregoing analysis the H1 and H2 loops aretreated separately and each compared against its closest matching humancanonical structural class)

The foregoing analysis relies on prediction of the canonical structureof the H1 and H2 loops of the antibody of interest. If the actualstructures of the H1 and H2 loops in the antibody of interest are known,for example based on X-ray crystallography, then the H1 and H2 loops inthe antibody of interest may also be scored as having a canonical foldstructure “substantially identical” to a canonical fold structure knownto occur in human antibodies if the length of the loop differs from thatof the closest matching human canonical structural class (typically by±1 or ±2 amino acids) but the actual structure of the H1 and H2 loops inthe antibody of interest matches the structure of a human canonicalfold.

Key amino acid residues found in the human canonical structural classesfor the first and second hypervariable loops of human VH domains (H1 andH2) are described by Chothia et al., J. Mol. Biol. 227:799-817 (1992),the contents of which are incorporated herein in their entirety byreference. In particular, Table 3 on page 802 of Chothia et al., whichis specifically incorporated herein by reference, lists preferred aminoacid residues at key sites for H1 canonical structures found in thehuman germline, whereas Table 4 on page 803, also specificallyincorporated by reference, lists preferred amino acid residues at keysites for CDR H2 canonical structures found in the human germline.

In one embodiment, both H1 and H2 in the VH domain of the antibody withhigh human homology exhibit a predicted or actual canonical foldstructure which is substantially identical to a canonical fold structurewhich occurs in human antibodies.

Antibodies with high human homology may comprise a VH domain in whichthe hypervariable loops H1 and H2 form a combination of canonical foldstructures which is identical to a combination of canonical structuresknown to occur in at least one human germline VH domain. It has beenobserved that only certain combinations of canonical fold structures atH1 and H2 actually occur in VH domains encoded by the human germline. Inan embodiment H1 and H2 in the VH domain of the antibody with high humanhomology may be obtained from a VH domain of a non-human species, e.g. aCamelidae species, yet form a combination of predicted or actualcanonical fold structures which is identical to a combination ofcanonical fold structures known to occur in a human germline orsomatically mutated VH domain. In non-limiting embodiments H1 and H2 inthe VH domain of the antibody with high human homology may be obtainedfrom a VH domain of a non-human species, e.g. a Camelidae species, andform one of the following canonical fold combinations: 1-1, 1-2, 1-3,1-6, 1-4, 2-1, 3-1 and 3-5.

An antibody with high human homology may contain a VH domain whichexhibits both high sequence identity/sequence homology with human VH,and which contains hypervariable loops exhibiting structural homologywith human VH.

It may be advantageous for the canonical folds present at H1 and H2 inthe VH domain of the antibody with high human homology, and thecombination thereof, to be “correct” for the human VH germline sequencewhich represents the closest match with the VH domain of the antibodywith high human homology in terms of overall primary amino acid sequenceidentity. By way of example, if the closest sequence match is with ahuman germline VH3 domain, then it may be advantageous for H1 and H2 toform a combination of canonical folds which also occurs naturally in ahuman VH3 domain. This may be particularly important in the case ofantibodies with high human homology which are derived from non-humanspecies, e.g. antibodies containing VH and VL domains which are derivedfrom camelid conventional antibodies, especially antibodies containinghumanised camelid VH and VL domains.

Thus, in one embodiment the VH domain of the CD70 antibody with highhuman homology may exhibit a sequence identity or sequence homology of80% or greater, 85% or greater, 90% or greater, 95% or greater, 97% orgreater, or up to 99% or even 100% with a human VH domain across theframework regions FR1, FR2, FR3 and FR4, and in addition H1 and H2 inthe same antibody are obtained from a non-human VH domain (e.g. derivedfrom a Camelidae species), but form a combination of predicted or actualcanonical fold structures which is the same as a canonical foldcombination known to occur naturally in the same human VH domain.

In other embodiments, L1 and L2 in the VL domain of the antibody withhigh human homology are each obtained from a VL domain of a non-humanspecies (e.g. a camelid-derived VL domain), and each exhibits apredicted or actual canonical fold structure which is substantiallyidentical to a canonical fold structure which occurs in humanantibodies.

As with the VH domains, the hypervariable loops of VL domains of bothVLambda and VKappa types can adopt a limited number of conformations orcanonical structures, determined in part by length and also by thepresence of key amino acid residues at certain canonical positions.

Within an antibody of interest having high human homology, L1, L2 and L3loops obtained from a VL domain of a non-human species, e.g. a Camelidaespecies, may be scored as having a canonical fold structure“substantially identical” to a canonical fold structure known to occurin human antibodies if at least the first, and preferable both, of thefollowing criteria are fulfilled:

1. An identical length, determined by the number of residues, to theclosest matching human structural class.

2. At least 33% identity, preferably at least 50% identity with the keyamino acid residues described for the corresponding human L1 or L2canonical structural classes, from either the VLambda or the VKapparepertoire.

(note for the purposes of the foregoing analysis the L1 and L2 loops aretreated separately and each compared against its closest matching humancanonical structural class)

The foregoing analysis relies on prediction of the canonical structureof the L1, L2 and L3 loops in the VL domain of the antibody of interest.If the actual structure of the L1, L2 and L3 loops is known, for examplebased on X-ray crystallography, then L1, L2 or L3 loops derived from theantibody of interest may also be scored as having a canonical foldstructure “substantially identical” to a canonical fold structure knownto occur in human antibodies if the length of the loop differs from thatof the closest matching human canonical structural class (typically by±1 or ±2 amino acids) but the actual structure of the Camelidae loopsmatches a human canonical fold.

Key amino acid residues found in the human canonical structural classesfor the CDRs of human VLambda and VKappa domains are described by Moreaet al. Methods, 20: 267-279 (2000) and Martin et al., J. Mol. Biol.,263:800-815 (1996). The structural repertoire of the human VKappa domainis also described by Tomlinson et al. EMBO J. 14:4628-4638 (1995), andthat of the VLambda domain by Williams et al. J. Mol. Biol., 264:220-232(1996). The contents of all these documents are to be incorporatedherein by reference.

L1 and L2 in the VL domain of an antibody with high human homology mayform a combination of predicted or actual canonical fold structureswhich is identical to a combination of canonical fold structures knownto occur in a human germline VL domain. In non-limiting embodiments L1and L2 in the VLambda domain of an antibody with high human homology(e.g. an antibody containing a camelid-derived VL domain or a humanisedvariant thereof) may form one of the following canonical foldcombinations: 11-7, 13-7(A,B,C), 14-7(A,B), 12-11, 14-11 and 12-12 (asdefined in Williams et al. J. Mol. Biol. 264:220-32 (1996) and as shownon http://www.bioc.uzh.ch/antibody/Sequences/Germlines/VBase_hVL.html).In non-limiting embodiments L1 and L2 in the Vkappa domain may form oneof the following canonical fold combinations: 2-1, 3-1, 4-1 and 6-1 (asdefined in Tomlinson et al. EMBO J. 14:4628-38 (1995) and as shown onhttp://www.bioc.uzh.ch/antibody/Sequences/Germlines/VBase_hVK.html).

In a further embodiment, all three of L1, L2 and L3 in the VL domain ofan antibody with high human homology may exhibit a substantially humanstructure. It is preferred that the VL domain of the antibody with highhuman homology exhibits both high sequence identity/sequence homologywith human VL, and also that the hypervariable loops in the VL domainexhibit structural homology with human VL.

In one embodiment, the VL domain of a CD70 antibody with high humanhomology may exhibit a sequence identity of 80% or greater, 85% orgreater, 90% or greater, 95% or greater, 97% or greater, or up to 99% oreven 100% with a human VL domain across the framework regions FR1, FR2,FR3 and FR4, and in addition hypervariable loop L1 and hypervariableloop L2 may form a combination of predicted or actual canonical foldstructures which is the same as a canonical fold combination known tooccur naturally in the same human VL domain.

It is, of course, envisaged that VH domains exhibiting high sequenceidentity/sequence homology with human VH, and also structural homologywith hypervariable loops of human VH will be combined with VL domainsexhibiting high sequence identity/sequence homology with human VL, andalso structural homology with hypervariable loops of human VL to provideantibodies with high human homology containing VH/VL pairings (e.gcamelid-derived VH/Vl pairings) with maximal sequence and structuralhomology to human-encoded VH/VL pairings.

As summarised above, the invention relates, at least in part, toantibodies, and antigen binding fragments thereof, that bind to humanCD70 with high affinity. The properties and characteristics of the CD70antibodies, and antibody fragments, according to the invention will nowbe described in further detail.

CD70 Binding and Affinity

In certain aspects, the antibodies, and antigen binding fragmentsthereof, provided herein bind to human CD70 with high affinity.Antibodies, or antigen binding fragments thereof, which bind to humanCD70 with high affinity may exhibit a binding affinity (K_(D)) for humanCD70, and more particularly the extracellular domain of human CD70, ofabout 10 nM or less, or 1 nM or less, or 0.1 nM or less, or 10 pM orless.

The CD70 antibody (or antigen binding fragment) may comprise VH and VLdomains which, when the VH and VL domains are expressed in the form of aFab, exhibit a dissociation off-rate for human CD70 binding of less than7×10⁻⁴ s⁻¹, preferably less than 5×10⁻⁴ s⁻¹, or less than 2×10⁻⁴ s⁻¹,less than 1×10⁻⁴ s⁻¹. Typically the off rate will fall in the range offrom 0.4×10⁻⁴ s⁻¹ to 4.8×10⁻⁴ s⁻¹. Binding affinity (K_(D)) anddissociation rate (K_(off)) can be measured using standard techniqueswell known to persons skilled in the art, such as for example surfaceplasmon resonance (BIAcore), as described in the accompanying examples.In brief, 50 μl of sample to be tested is added to 200 μl PBS+0.02%Tween, and diluted 1/400 as follows: 5 μl of sample (1 mg/ml)+195 μlHBS-EP+(Biacore buffer), further diluted 10 μl+90 μl HVS-EP+=2.5 μg/ml.Biacore analysis is performed at room temperature using a highly CD70coated CM5 chip (4000 RU) using the manufacturer's supplied protocol.

In this regard, it should be noted that although the off-rate ismeasured for VH and VL combinations in the form of Fabs, this does notmean that the VH and VL domains contribute equally to CD70 binding. Formany VH/VL combinations, it is the VH domain which mainly contributes toCD70 binding, with the VL domain contributing to solubility and/orstability of the VH/VL pairing.

Binding of the CD70 antibodies described herein, or Fab fragmentsthereof, to recombinant human CD70 may also be assessed by ELISA.

The CD70 antibodies described herein may further exhibit binding to CD70expressed on the surface of intact cells, e.g. 786-O renal carcinomacells and other cancer cell lines. Binding of CD70 antibodies toCD70-expressing cells may be assessed by flow cytometry. The resultspresented in the accompanying examples demonstrate that preferred CD70antibodies, including variant 41D12 (when expressed as non-fucosylatedIgG1 ARGX-110), exhibit very strong binding to cell-surface CD70.Indeed, for many cell lines ARGX-110 exhibits stronger binding thancomparator prior art antibody SGN70.

It is particularly noteworthy that ARGX-110 exhibits strong binding tocell lines which express CD70 at low copy number, including inter aliathe cell lines Raji, SU-DHL-6, MHHPREB1, Mino, Mec1, JVM-2, HH andEBC-1, and also to cancer cells isolated from patients (e.g. CLLpatients), in both cases the binding of ARGX-110 is markedly strongerthan SGN70. The “improved” binding to low copy number cell lines and CLLpatient materials may in large part reflect the extremely high affinityof ARGX-110 for recombinant CD70. These binding characteristics aresupportive of a particular utility of ARGX-110 (based on 41D12) andother CD70 antibodies described herein with similar properties in cancertreatment, particularly when utilised as an IgG exhibiting potenteffector function (e.g. non-fucosylated IgG1) rather than as animmunoconjugate linked to a cytotoxic or cytostatic moiety. Thesignificance of high affinity binding (i.e. higher affinity than can beachieved with prior art CD70 antibodies) to recombinant CD70 and cellsurface CD70 with regard to clinical utility of the antibodies isdemonstrated in the accompanying examples by experiments in which PBMCsamples are “spiked” with cancer cell lines and then treated with CD70antibodies. In this system treatment with ARGX-110 producedsignificantly more lysis of the target cancer cells than the comparatorCD70 antibodies MDX1411 and SGN70.

The CD70 antibodies described herein exhibit high affinity binding tohuman CD70, and more specifically the extracellular domain of humanCD70, but cross-reactivity with non-human homologues of CD70 is notexcluded. Indeed, it is an advantageous feature of many of the CD70antibodies described herein, including 27B3 and its germlined variants,particularly 41D12, that in addition to high affinity binding to humanCD70 they also cross-react with simian CD70 homologs, specifically theCD70 homologs of rhesus macaque and cynomolgus monkey.

In this regard, a CD70 antibody can be considered to exhibitcross-reactivity with a simian CD70 homolog, for example the CD70homologs of rhesus macaque and cynomolgus monkey, if the difference inIC50 for human versus simian (rhesus or cynomolgus monkey) CD70 in aCD70-CD27 inhibition ELISA, such as that described in the accompanyingexample 20.2, is less than 5-fold, preferably less than 3-fold or lessthan 2-fold. The binding affinity, and therefore blocking potency, forhuman and simian CD70 should be broadly comparable.

Interaction Between CD70 and CD27

In certain aspects, the CD70 antibodies, or antigen binding fragmentsthereof, provided herein may bind to human CD70 with high affinity andblock the interaction between CD70 and CD27.

The ability of CD70 antibodies, or antigen binding fragments thereof, toblock binding of CD70 to CD27 may be assessed by Elisa using eithercaptured recombinant CD70 (Flag-TNC-CD70) or directed coated CD70 andrecombinant CD27-Fc. The CD70 antibodies described herein may inhibitthe interaction between CD70 and CD27 with an IC50 of 300 ng/ml or less,or 200 ng/ml or less, or 110 ng/ml or less, or 70 ng/ml or less, or 50ng/ml or less.

The ability of CD70 antibodies, or antigen binding fragments thereof, toblock binding of CD70 to CD27 may also be assessed in an assay based onco-culture of Raji cells (human B cell lymphoma) and HT1080-CD27 cells(human epithelial cell line transfected with CD27), as described in theaccompanying examples. The CD70 antibodies described which inhibit theinteraction between CD70 and CD27 may exhibit an IC50 in this co-cultureassay of 500 ng/ml or less, or 300 ng/ml or less, or 100 ng/ml or less,or 50 ng/ml or less, or 30 ng/ml or less.

The preferred CD70 antibodies based on 27B3 and germlined variantsthereof, including 41D12 (ARGX-110) exhibit potent blocking of theCD70/CD27 interaction in the Elisa system. Indeed, ARGX-110 issignificantly more potent in blocking CD70/CD27 interactions thancomparator antibodies SGN70 and MDX1411. The interaction between CD70and CD27 may contribute to tumour cell survival, proliferation and/orimmune suppression within the tumour microenvironment. Accordingly,potent inhibition of the CD70/CD27 interaction, in addition to highaffinity binding to CD70, may contribute to improved clinical outcome inthe treatment of certain CD70-expressing cancers or immunologicaldisorders, for example autoimmune diseases and cancers which co-expressCD70 and CD27.

In other embodiments, the CD70 antibodies, or antigen binding fragmentsthereof, may bind to human CD70 but not inhibit the interaction betweenCD70 and CD27. CD70 antibodies which bind CD70 but do not inhibit theinteraction between CD70 and CD27 can be assessed based on Elisa usingeither captured recombinant CD70 (Flag-TNC-CD70) or directed coated CD70and recombinant CD27-Fc. As demonstrated in the accompanying examples, anumber of Fabs have been identified which bind CD70 but do not inhibitthe CD70/CD27 interaction to a significant extent. In particular, theFab clone identified herein as 59D10 exhibits strong binding torecombinant CD70 as measured by Biacore but does not block the CD70/CD27interaction to a significant extent, when assessed by ELISA. CD70antibodies described herein which bind CD70 but do not inhibit theinteraction between CD70 and CD27 may still possess intact antibodyeffector functions, i.e. one or more of ADCC, CDC, ADCP or ADC andinhibit tumour cell growth in vivo. CD70 antibodies which bind CD70 withhigh affinity but are non-blocking may be advantageously utilised as“one-armed” antibodies, or PEGylated Fab products or in any otherantibody format which provides a strict monovalent interaction with atarget cell expressing CD70.

CD70 Epitopes

In certain aspects, the CD70 antibodies described herein bind toepitopes within the extracellular domain of human CD70.

The term “epitope” refers to the portion(s) of an antigen (e.g. humanCD70) that contact an antibody. Epitopes can be linear, i.e., involvingbinding to a single sequence of amino acids, or conformational, i.e.,involving binding to two or more sequences of amino acids in variousregions of the antigen that may not necessarily be contiguous.

The CD70 antibodies provided herein may bind to different (overlappingor non-overlapping) epitopes within the extracellular domain of thehuman CD70 protein. For example, the Fabs denoted 1C2 and 9E1 in theaccompanying examples clearly bind to different, non-overlappingepitopes on human CD70.

As noted elsewhere herein, the preferred CD70 antibody 41D12 (ARGX-110)exhibits a particularly useful combination of binding characteristicswhich is not exhibited by any known prior art CD70 antibody, namely:

-   -   (a) binding within the amino acid sequence HIQVTLAICSS (SEQ ID        NO:342) in human CD70;    -   (b) cross-reactivity with CD70 homologs of rhesus macaque        (Macaca mulatta) and cynomolgus monkey (Macaca cynomolgus);    -   (c) binding to native human CD70 and heat denatured recombinant        human CD70.

For any given CD70 antibody, the ability to bind human CD70 within theamino acid sequence HIQVTLAICSS (SEQ ID NO:342) can be readilydetermined by a person of ordinary skill in the art, for example usingthe mouse-human chimeric CD70 binding analysis described in theaccompanying example 20.4.

Cross-reactivity with simian CD70 homologs can also be readilydetermined by a person of ordinary skill in the art, for example by FACsanalysis, surface plasmon resonance (Biacore™) or using a CD70-CD27inhibition ELISA. In this regard, a CD70 antibody can be considered toexhibit cross-reactivity with a simian CD70 homolog, for example theCD70 homologs of rhesus macaque and cynomolgus monkey, if the differencein IC50 for human versus simian (rhesus or cynomolgus monkey) CD70 in aCD70-CD27 inhibition ELISA, such as that described in the accompanyingexample 20.2, is less than 5-fold, preferably less than 3-fold or lessthan 2-fold.

The CD70 antibodies described herein may also demonstrate the ability tobind both native human CD70 and heat denatured recombinant human CD70.In this connection, binding to “native” human CD70 is indicated bybinding to CD70 expressed on the surface of a CD70-expressing cell, suchas any of the CD70+ cell lines described in the accompanying examples,or even natural CD70-expressing cells isolated from patient material(e.g. cells isolated from CLL patients as in the accompanying examples),activated T cells, etc. Binding to “native” human CD70 can thus beeasily tested by a person of ordinary skill in the art.

Binding to heat denatured recombinant human CD70 can be tested asdescribed in the accompanying example 20.3. It is preferred that theCD70 antibody should exhibit an OD at 620 nm of 0.6 or greater in thisassay, in order to demonstrate significant binding to heat denaturedrecombinant human CD70.

Partial or Slow Internalisation

The CD70 antibodies described herein, including the preferred CD70antibody 41D12 (ARGX-110), exhibit partial internalisation in renalcarcinoma cell lines, meaning that when antibody internalisation istested in 786-O renal carcinoma cells a substantial proportion of thebound antibody, i.e. at least 30-40%, remains external after 6 hours,and even after 24 hours, incubation at 37° C. In addition, the CD70antibodies may also exhibit a slow rate of internalisation. In thisregard, a CD70 antibody is considered to exhibit slow internalisation ifit is internalised at a slower rate than a reference CD70 antibody 9D1(VH SEQ ID NO:178; VL SEQ ID NO:190).

As demonstrated in the accompanying examples, different cancer celllines exhibit significant differences in the degree of internalisationof CD70 antibodies. It is particularly significant that the majority ofcancer cell lines exhibit less than 30%, and even less than 10%,internalisation of bound CD70 antibody, even after 6 hours incubationwith ARGX-110. These results are clearly supportive of the utility ofCD70 antibodies with potent effector function (including variantsengineered for enhanced effector function) in the treatment ofCD70-expressing cancers. It has previously been reported in thescientific literature that CD70 is an “internalising” target; hence ithas been proposed to develop CD70 antibody-drug immunoconjugates fortreatment of both CD70-expressing cancers and immunological diseases.Therefore, the results presented herein, which conclusively demonstratethat internalisation of cell-surface bound CD70 antibodies is a rareevent and that the majority of CD70-expressing cell lines do notinternalise bound CD70 antibody to a significant extent, are extremelysurprising.

Camelid-Derived CD70 Antibodies

In yet other aspects, the antibodies or antigen binding fragmentsthereof described herein may comprise at least one hypervariable loop orcomplementarity determining region obtained from a VH domain or a VLdomain of a species in the family Camelidae. In particular, the antibodyor antigen binding fragment may comprise VH and/or VL domains, or CDRsthereof, obtained by active immunisation of outbred camelids, e.g.llamas, with a human CD70 antigen.

By “hypervariable loop or complementarity determining region obtainedfrom a VH domain or a VL domain of a species in the family Camelidae” ismeant that that hypervariable loop (HV) or CDR has an amino acidsequence which is identical, or substantially identical, to the aminoacid sequence of a hypervariable loop or CDR which is encoded by aCamelidae immunoglobulin gene. In this context “immunoglobulin gene”includes germline genes, immunoglobulin genes which have undergonerearrangement, and also somatically mutated genes. Thus, the amino acidsequence of the HV or CDR obtained from a VH or VL domain of a Camelidaespecies may be identical to the amino acid sequence of a HV or CDRpresent in a mature Camelidae conventional antibody. The term “obtainedfrom” in this context implies a structural relationship, in the sensethat the HVs or CDRs of the CD70 antibody embody an amino acid sequence(or minor variants thereof) which was originally encoded by a Camelidaeimmunoglobulin gene. However, this does not necessarily imply aparticular relationship in terms of the production process used toprepare the CD70 antibody.

Camelid-derived CD70 antibodies may be derived from any camelid species,including inter alia, llama, dromedary, alpaca, vicuna, guanaco orcamel.

CD70 antibodies comprising camelid-derived VH and VL domains, or CDRsthereof, are typically recombinantly expressed polypeptides, and may bechimeric polypeptides. The term “chimeric polypeptide” refers to anartificial (non-naturally occurring) polypeptide which is created byjuxtaposition of two or more peptide fragments which do not otherwiseoccur contiguously. Included within this definition are “species”chimeric polypeptides created by juxtaposition of peptide fragmentsencoded by two or more species, e.g. camelid and human.

Camelid-derived CDRs may comprise one of the CDR sequences shown as SEQID Nos: 49-59, 262 or 263 (heavy chain CDR3), or SEQ ID Nos: 26-37, 249,258 or 259 (heavy chain CDR2) or SEQ ID Nos: 10-20, 248, 256 or 257(heavy chain CDR1) or one of the CDR sequences shown as SEQ ID NOs:148-168, 271 or 273 (light chain CDR3), or SEQ ID Nos: 109-128 or 270(light chain CDR2) or SEQ ID Nos:77-95, or 250-253, 267 and 268 (lightchain CDR1).

In one embodiment the entire VH domain and/or the entire VL domain maybe obtained from a species in the family Camelidae. In specificembodiments, the camelid-derived VH domain may comprise the amino acidsequence shown as SEQ ID NOs: 177-188, 212-223, 274 or 275, whereas thecamelid-derived VL domain may comprise the amino acid sequence show asSEQ ID Nos:189-211, 230-245, 276 or 277 (VL). The camelid-derived VHdomain and/or the camelid-derived VL domain may then be subject toprotein engineering, in which one or more amino acid substitutions,insertions or deletions are introduced into the camelid amino acidsequence. These engineered changes preferably include amino acidsubstitutions relative to the camelid sequence. Such changes include“humanisation” or “germlining” wherein one or more amino acid residuesin a camelid-encoded VH or VL domain are replaced with equivalentresidues from a homologous human-encoded VH or VL domain.

Isolated camelid VH and VL domains obtained by active immunisation of acamelid (e.g. llama) with a human CD70 antigen can be used as a basisfor engineering antigen binding polypeptides according to the invention.Starting from intact camelid VH and VL domains, it is possible toengineer one or more amino acid substitutions, insertions or deletionswhich depart from the starting camelid sequence. In certain embodiments,such substitutions, insertions or deletions may be present in theframework regions of the VH domain and/or the VL domain. The purpose ofsuch changes in primary amino acid sequence may be to reduce presumablyunfavourable properties (e.g. immunogenicity in a human host (so-calledhumanization), sites of potential product heterogeneity and orinstability (glycosylation, deamidation, isomerisation, etc.) or toenhance some other favourable property of the molecule (e.g. solubility,stability, bioavailability, etc.). In other embodiments, changes inprimary amino acid sequence can be engineered in one or more of thehypervariable loops (or CDRs) of a Camelidae VH and/or VL domainobtained by active immunisation. Such changes may be introduced in orderto enhance antigen binding affinity and/or specificity, or to reducepresumably unfavourable properties, e.g. immunogenicity in a human host(so-called humanization), sites of potential product heterogeneity andor instability, glycosylation, deamidation, isomerisation, etc., or toenhance some other favourable property of the molecule, e.g. solubility,stability, bioavailability, etc.

Thus, in one embodiment, the invention provides a variant CD70 antibodywhich contains at least one amino acid substitution in at least oneframework or CDR region of either the VH domain or the VL domain incomparison to a camelid-derived VH or VL domain, examples of whichinclude but are not limited to the camelid VH domains comprising theamino acid sequences shown as SEQ ID NO: 177-188, 212-223, 274 or 275,and the camelid VL domains comprising the amino acid sequences show asSEQ ID NO: 189-211, 230-245, 276 or 277.

In other embodiments, there are provided “chimeric” antibody moleculescomprising camelid-derived VH and VL domains (or engineered variantsthereof) and one or more constant domains from a non-camelid antibody,for example human-encoded constant domains (or engineered variantsthereof). In such embodiments it is preferred that both the VH domainand the VL domain are obtained from the same species of camelid, forexample both VH and VL may be from Lama glama or both VH and VL may befrom Lama pacos (prior to introduction of engineered amino acid sequencevariation). In such embodiments both the VH and the VL domain may bederived from a single animal, particularly a single animal which hasbeen actively immunised with a human CD70 antigen.

As an alternative to engineering changes in the primary amino acidsequence of Camelidae VH and/or VL domains, individual camelid-derivedhypervariable loops or CDRs, or combinations thereof, can be isolatedfrom camelid VH/VL domains and transferred to an alternative (i.e.non-Camelidae) framework, e.g. a human VH/VL framework, by CDR grafting.In particular, non-limiting, embodiments the camelid-derived CDRs may beselected from CDRs having the amino acid sequences shown as SEQ ID NOs:49-59 (heavy chain CDR3), or SEQ ID Nos: 26-37 (heavy chain CDR2) or SEQID Nos: 10-20 (heavy chain CDR1) or one of the CDR sequences shown asSEQ ID NOs: 148-168 (light chain CDR3), or SEQ ID Nos: 109-128 (lightchain CDR2) or SEQ ID Nos:77-95 (light chain CDR1).

CD70 antibodies comprising camelid-derived VH and VL domains, or CDRsthereof, can take various different embodiments in which both a VHdomain and a VL domain are present. The term “antibody” herein is usedin the broadest sense and encompasses, but is not limited to, monoclonalantibodies (including full length monoclonal antibodies), polyclonalantibodies, multispecific antibodies (e.g., bispecific antibodies), solong as they exhibit the appropriate immunological specificity for ahuman CD70 protein. The term “monoclonal antibody” as used herein refersto an antibody obtained from a population of substantially homogeneousantibodies, i.e., the individual antibodies comprising the populationare identical except for possible naturally occurring mutations that maybe present in minor amounts. Monoclonal antibodies are highly specific,being directed against a single antigenic site. Furthermore, in contrastto conventional (polyclonal) antibody preparations which typicallyinclude different antibodies directed against different determinants(epitopes) on the antigen, each monoclonal antibody is directed againsta single determinant or epitope on the antigen.

“Antibody fragments” comprise a portion of a full length antibody,generally the antigen binding or variable domain thereof. Examples ofantibody fragments include Fab, Fab′, F(ab′)2, bi-specific Fab′ s, andFv fragments, diabodies, linear antibodies, single-chain antibodymolecules, a single chain variable fragment (scFv) and multispecificantibodies formed from antibody fragments (see Holliger and Hudson,Nature Biotechnol. 23:1126-36 (2005), the contents of which areincorporated herein by reference).

In non-limiting embodiments, CD70 antibodies comprising camelid-derivedVH and VL domains, or CDRs thereof, may comprise CH1 domains and/or CLdomains, the amino acid sequence of which is fully or substantiallyhuman. Where the antigen binding polypeptide of the invention is anantibody intended for human therapeutic use, it is typical for theentire constant region of the antibody, or at least a part thereof, tohave fully or substantially human amino acid sequence. Therefore, one ormore or any combination of the CH1 domain, hinge region, CH2 domain, CH3domain and CL domain (and CH4 domain if present) may be fully orsubstantially human with respect to it's amino acid sequence.

Advantageously, the CH1 domain, hinge region, CH2 domain, CH3 domain andCL domain (and CH4 domain if present) may all have fully orsubstantially human amino acid sequence. In the context of the constantregion of a humanised or chimeric antibody, or an antibody fragment, theterm “substantially human” refers to an amino acid sequence identity ofat least 90%, or at least 92%, or at least 95%, or at least 97%, or atleast 99% with a human constant region. The term “human amino acidsequence” in this context refers to an amino acid sequence which isencoded by a human immunoglobulin gene, which includes germline,rearranged and somatically mutated genes. The invention alsocontemplates polypeptides comprising constant domains of “human”sequence which have been altered, by one or more amino acid additions,deletions or substitutions with respect to the human sequence, exceptingthose embodiments where the presence of a “fully human” hinge region isexpressly required.

The presence of a “fully human” hinge region in the CD70 antibodies ofthe invention may be beneficial both to minimise immunogenicity and tooptimise stability of the antibody.

As discussed elsewhere herein, it is contemplated that one or more aminoacid substitutions, insertions or deletions may be made within theconstant region of the heavy and/or the light chain, particularly withinthe Fc region. Amino acid substitutions may result in replacement of thesubstituted amino acid with a different naturally occurring amino acid,or with a non-natural or modified amino acid. Other structuralmodifications are also permitted, such as for example changes inglycosylation pattern (e.g. by addition or deletion of N- or O-linkedglycosylation sites). Depending on the intended use of the antibody, itmay be desirable to modify the antibody of the invention with respect toits binding properties to Fc receptors, for example to modulate effectorfunction. For example cysteine residue(s) may be introduced in the Fcregion, thereby allowing interchain disulfide bond formation in thisregion. The homodimeric antibody thus generated may have improvedinternalization capability and/or increased complement-mediated cellkilling and antibody-dependent cellular cytotoxicity (ADCC). See Caronet al., J. Exp. Med. 176:1191-1195 (1992) and Shopes, B. J. Immunol.148:2918-2922 (1992). Alternatively, a CD70 antibody can be engineeredwhich has dual Fc regions and may thereby have enhanced complement lysisand ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design3:219-230 (1989). The invention also contemplates immunoconjugatescomprising an antibody as described herein conjugated to a cytotoxicagent such as a chemotherapeutic agent, toxin (e.g., an enzymaticallyactive toxin of bacterial, fungal, plant or animal origin, or fragmentsthereof), or a radioactive isotope (i.e., a radioconjugate). Fc regionsmay also be engineered for half-life extension, as described by Chan andCarter, Nature Reviews: Immunology, Vol. 10, pp 301-316, 2010,incorporated herein by reference.

In yet another embodiment, the Fc region is modified to increase theability of the antibody to mediate antibody dependent cellularcytotoxicity (ADCC) and/or to increase the affinity of the antibody foran Fcγ receptor by modifying one or more amino acids.

In still another embodiment, the glycosylation of an antibody ismodified. For example, an aglycoslated antibody can be made (i.e., theantibody lacks glycosylation). Glycosylation can be altered to, forexample, increase the affinity of the antibody for the CD70 targetantigen. Such carbohydrate modifications can be accomplished by; forexample, altering one or more sites of glycosylation within the antibodysequence. For example, one or more amino acid substitutions can be madethat result in elimination of one or more variable region frameworkglycosylation sites to thereby eliminate glycosylation at that site.Such aglycosylation may increase the affinity of the antibody forantigen.

Also envisaged are variant CD70 antibodies having an altered type ofglycosylation, such as a hypofucosylated antibody having reduced amountsof fucosyl residues or a non-fucosylated antibody (as described byNatsume et al., Drug Design Development and Therapy, Vol. 3, pp 7-16,2009) or an antibody having increased bisecting GlcNac structures. Suchaltered glycosylation patterns have been demonstrated to increase theADCC activity of antibodies, producing typically 10-fold enhancement ofADCC relative to an equivalent antibody comprising a “native” human Fcdomain. Such carbohydrate modifications can be accomplished by, forexample, expressing the antibody in a host cell with alteredglycosylation enzymatic machinery (as described by Yamane-Ohnuki andSatoh, mAbs 1:3, 230-236, 2009). Examples of non-fucosylated antibodieswith enhanced ADCC function are those produced using the Potelligent™technology of BioWa Inc.

The invention can, in certain embodiments, encompass chimericCamelidae/human antibodies, and in particular chimeric antibodies inwhich the VH and VL domains are of fully camelid sequence (e.g. Llama oralpaca) and the remainder of the antibody is of fully human sequence.CD70 antibodies can include antibodies comprising “humanised” or“germlined” variants of camelid-derived VH and VL domains, or CDRsthereof, and camelid/human chimeric antibodies, in which the VH and VLdomains contain one or more amino acid substitutions in the frameworkregions in comparison to camelid VH and VL domains obtained by activeimmunisation of a camelid with a human CD70 antigen. Such “humanisation”increases the % sequence identity with human germline VH or VL domainsby replacing mis-matched amino acid residues in a starting Camelidae VHor VL domain with the equivalent residue found in a humangermline-encoded VH or VL domain.

CD70 antibodies may also be CDR-grafted antibodies in which CDRs (orhypervariable loops) derived from a camelid antibody, for example ancamelid CD70 antibody raised by active immunisation with human CD70protein, or otherwise encoded by a camelid gene, are grafted onto ahuman VH and VL framework, with the remainder of the antibody also beingof fully human origin. Such CDR-grafted CD70 antibodies may contain CDRshaving the amino acid sequences shown as SEQ ID Nos: 49-59, 262 or 263(heavy chain CDR3), or SEQ ID Nos: 26-37, 249, 258 or 259 (heavy chainCDR2) or SEQ ID Nos: 10-20, 248, 256 or 257 (heavy chain CDR1) or one ofthe CDR sequences shown as SEQ ID NOs: 148-168, 271 or 273 (light chainCDR3), or SEQ ID Nos: 109-128 or 270 (light chain CDR2) or SEQ IDNos:77-95, or 250-253, 267 and 268 (light chain CDR1).

Humanised, chimeric and CDR-grafted CD70 antibodies as described above,particularly antibodies comprising hypervariable loops or CDRs derivedfrom active immunisation of camelids with a human CD70 antigen, can bereadily produced using conventional recombinant DNA manipulation andexpression techniques, making use of prokaryotic and eukaryotic hostcells engineered to produce the polypeptide of interest and includingbut not limited to bacterial cells, yeast cells, mammalian cells, insectcells, plant cells, some of them as described herein and illustrated inthe accompanying examples.

Camelid-derived CD70 antibodies include variants wherein thehypervariable loop(s) or CDR(s) of the VH domain and/or the VL domainare obtained from a conventional camelid antibody raised against humanCD70, but wherein at least one of said (camelid-derived) hypervariableloops or CDRs has been engineered to include one or more amino acidsubstitutions, additions or deletions relative to the camelid-encodedsequence. Such changes include “humanisation” of the hypervariableloops/CDRs. Camelid-derived HVs/CDRs which have been engineered in thismanner may still exhibit an amino acid sequence which is “substantiallyidentical” to the amino acid sequence of a camelid-encoded HV/CDR. Inthis context, “substantial identity” may permit no more than one, or nomore than two amino acid sequence mis-matches with the camelid-encodedHV/CDR. Particular embodiments of the CD70 antibody may containhumanised variants of the CDR sequences shown as SEQ ID Nos: 49-59, 262or 263 (heavy chain CDR3), or SEQ ID Nos: 26-37, 249, 258 or 259 (heavychain CDR2) or SEQ ID Nos: 10-20, 248, 256 or 257 (heavy chain CDR1) orone of the CDR sequences shown as SEQ ID NOs: 148-168, 271 or 273 (lightchain CDR3), or SEQ ID Nos: 109-128 or 270 (light chain CDR2) or SEQ IDNos:77-95, or 250-253, 267 and 268 (light chain CDR1).

The camelid-derived CD70 antibodies provided herein may be of anyisotype. Antibodies intended for human therapeutic use will typically beof the IgA, IgD, IgE IgG, IgM type, often of the IgG type, in which casethey can belong to any of the four sub-classes IgG1, IgG2a and b, IgG3or IgG4. Within each of these sub-classes it is permitted to make one ormore amino acid substitutions, insertions or deletions within the Fcportion, or to make other structural modifications, for example toenhance or reduce Fc-dependent functionalities.

Humanisation of Camelid-Derived VH and VL Domains

Camelid conventional antibodies provide an advantageous starting pointfor the preparation of antibodies with utility as human therapeuticagents due to the following factors, discussed in U.S. Ser. No.12/497,239 which is incorporated herein by reference:

1) High % sequence homology between camelid VH and VL domains and theirhuman counterparts;

2) High degree of structural homology between CDRs of camelid VH and VLdomains and their human counterparts (i.e. human-like canonical foldstructures and human-like combinations of canonical folds).

The camelid (e.g. llama) platform also provides a significant advantagein terms of the functional diversity of the CD70 antibodies which can beobtained.

The utility of CD70 antibodies comprising camelid VH and/or camelid VLdomains for human therapy can be improved still further by“humanisation” of natural camelid VH and VL domains, for example torender them less immunogenic in a human host. The overall aim ofhumanisation is to produce a molecule in which the VH and VL domainsexhibit minimal immunogenicity when introduced into a human subject,whilst retaining the specificity and affinity of the antigen bindingsite formed by the parental VH and VL domains.

One approach to humanisation, so-called “germlining”, involvesengineering changes in the amino acid sequence of a camelid VH or VLdomain to bring it closer to the germline sequence of a human VH or VLdomain.

Determination of homology between a camelid VH (or VL) domain and humanVH (or VL) domains is a critical step in the humanisation process, bothfor selection of camelid amino acid residues to be changed (in a givenVH or VL domain) and for selecting the appropriate replacement aminoacid residue(s).

An approach to humanisation of camelid conventional antibodies has beendeveloped based on alignment of a large number of novel camelid VH (andVL) domain sequences, typically somatically mutated VH (or VL) domainswhich are known to bind a target antigen, with human germline VH (or VL)sequences, human VH (and VL) consensus sequences, as well as germlinesequence information available for llama pacos. The following passagesoutline the principles which can be applied to (i) select “camelid”amino acid residues for replacement in a camelid-derived VH or VL domainor a CDR thereof, and (ii) select replacement “human” amino acidresidues to substitute in, when humanising any given camelid VH (or VL)domain. This approach can be used to prepare humanised variants ofcamelid-derived CDRs having the amino acid sequences shown as SEQ IDNos: 49-59, 262 or 263 (heavy chain CDR3), or SEQ ID Nos: 26-37, 249,258 or 259 (heavy chain CDR2) or SEQ ID Nos: 10-20, 248, 256 or 257(heavy chain CDR1) or one of the CDR sequences shown as SEQ ID NOs:148-168, 271 or 273 (light chain CDR3), or SEQ ID Nos: 109-128 or 270(light chain CDR2) or SEQ ID Nos:77-95, or 250-253, 267 or 268 (lightchain CDR1), and also for humanisation of camelid-derived VH domainshaving the sequences shown as SEQ ID NOs: 177-188, 212-223, 274 or 275and of camelid-derived VL domains having the sequences shown as SEQ IDNos:189-211, 230-245, 276 or 277.

Step 1. Select human (germline) family and member of this family thatshows highest homology/identity to the mature camelid sequence to behumanised. A general procedure for identifying the closest matchinghuman germline for any given camelid VH (or VL) domain is outlinedbelow.Step 2. Select specific human germline family member used to germlineagainst. Preferably this is the germline with the highest homology oranother germline family member from the same family.Step 3. Identify the preferred positions considered for germlining onthe basis of the table of amino acid utilisation for the camelidgermline that is closest to the selected human germline.Step 4. Try to change amino acids in the camelid germline that deviatefrom the closest human germline; germlining of FR residues is preferredover CDR residues.a. Preferred are positions that are deviating from the selected humangermline used to germline against, for which the amino acid found in thecamelid sequence does not match with the selected germline and is notfound in other germlines of the same subclass (both for V as well as forJ encoded FR amino acids).b. Positions that are deviating from the selected human germline familymember but which are used in other germlines of the same family may alsobe addressed in the germlining process.c. Additional mismatches (e.g. due to additional somatic mutations)towards the selected human germline may also be addressed.

The following approach may be used to determine the closest matchinghuman germline for a given camelid VH (or VL) domain:

Before analyzing the percentage sequence identity between Camelidae andhuman germline VH and VL, the canonical folds may first be determined,which allows the identification of the family of human germline segmentswith the identical combination of canonical folds for H1 and H2 or L1and L2 (and L3). Subsequently the human germline family member that hasthe highest degree of sequence homology with the Camelidae variableregion of interest may be chosen for scoring sequence homology. Thedetermination of Chothia canonical classes of hypervariable loops L1,L2, L3, H1 and H2 can be performed with the bioinformatics toolspublicly available on webpage www.bioinf.org.uk/abs/chothia.html.page.The output of the program shows the key residue requirements in adatafile. In these datafiles, the key residue positions are shown withthe allowed amino acids at each position. The sequence of the variableregion of the antibody is given as input and is first aligned with aconsensus antibody sequence to assign the Kabat numbering scheme. Theanalysis of the canonical folds uses a set of key residue templatesderived by an automated method developed by Martin and Thornton (Martinet al., J. Mol. Biol. 263:800-815 (1996)). The boundaries of theindividual framework regions may be assigned using the IMGT numberingscheme, which is an adaptation of the numbering scheme of Chothia(Lefranc et al., NAR 27: 209-212 (1999); http://imgt.cines.fr).

With the particular human germline V segment known, which uses the samecombination of canonical folds for H1 and H2 or L1 and L2 (and L3), thebest matching family member in terms of sequence homology can bedetermined. The percentage sequence identity between Camelidae VH and VLdomain framework amino acid sequences and corresponding sequencesencoded by the human germline can be determined using bioinformatictools, but manual alignment of the sequences could also be used. Humanimmunoglobulin sequences can be identified from several protein databases, such as VBase (http://vbase.mrc-cpe.cam.ac.uk/) or thePluckthun/Honegger database(http://www.bioc.unizh.ch/antibody/Sequences/Germlines. To compare thehuman sequences to the V regions of Camelidae VH or VL domains asequence alignment algorithm such as available via websites likewww.expasy.ch/tools/#align can be used, but also manual alignment canalso be performed with a limited set of sequences. Human germline lightand heavy chain sequences of the families with the same combinations ofcanonical folds and with the highest degree of homology with theframework regions 1, 2, and 3 of each chain may be selected and comparedwith the Camelidae variable region of interest; also the FR4 is checkedagainst the human germline JH and JK or JL regions.

Note that in the calculation of overall percent sequence homology theresidues of FR1, FR2 and FR3 are evaluated using the closest matchsequence from the human germline family with the identical combinationof canonical folds. Only residues different from the closest match orother members of the same family with the same combination of canonicalfolds are scored (NB—excluding any primer-encoded differences). However,for the purposes of humanization, residues in framework regionsidentical to members of other human germline families, which do not havethe same combination of canonical folds, can be considered forhumanization, despite the fact that these are scored “negative”according to the stringent conditions described above. This assumptionis based on the “mix and match” approach for humanization, in which eachof FR1, FR2, FR3 and FR4 is separately compared to its closest matchinghuman germline sequence and the humanized molecule therefore contains acombination of different FRs as was done by Qu and colleagues (Qu etla., Clin. Cancer Res. 5:3095-3100 (1999)) and Ono and colleagues (Onoet al., Mol. Immunol. 36:387-395 (1999)).

Cross-Competing Antibodies

Monoclonal antibodies or antigen-binding fragments thereof that“cross-compete” with the molecules disclosed herein are those that bindhuman CD70 at site(s) that are identical to, or overlapping with, thesite(s) at which the present CD70 antibodies bind. Competing monoclonalantibodies or antigen-binding fragments thereof can be identified, forexample, via an antibody competition assay. For example, a sample ofpurified or partially purified human CD70 can be bound to a solidsupport. Then, an antibody compound or antigen binding fragment thereofof the present invention and a monoclonal antibody or antigen-bindingfragment thereof suspected of being able to compete with such inventionantibody compound are added. One of the two molecules is labelled. Ifthe labelled compound and the unlabeled compound bind to separate anddiscrete sites on CD70, the labelled compound will bind to the samelevel whether or not the suspected competing compound is present.However, if the sites of interaction are identical or overlapping, theunlabeled compound will compete, and the amount of labelled compoundbound to the antigen will be lowered. If the unlabeled compound ispresent in excess, very little, if any, labelled compound will bind. Forpurposes of the present invention, competing monoclonal antibodies orantigen-binding fragments thereof are those that decrease the binding ofthe present antibody compounds to CD70 by about 50%, about 60%, about70%, about 80%, about 85%, about 90%, about 95%, or about 99%. Detailsof procedures for carrying out such competition assays are well known inthe art and can be found, for example, in Harlow and Lane (1988)Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., pages 567-569, ISBN 0-87969-314-2. Such assayscan be made quantitative by using purified antibodies. A standard curveis established by titrating one antibody against itself, i.e., the sameantibody is used for both the label and the competitor. The capacity ofan unlabeled competing monoclonal antibody or antigen-binding fragmentthereof to inhibit the binding of the labelled molecule to the plate istitrated. The results are plotted, and the concentrations necessary toachieve the desired degree of binding inhibition are compared.

Preferred embodiments are antibodies which cross-compete for binding tohuman CD70 with antibodies comprising the llama-derived Fab 27B3, andits germlined variants, including in particular 41D12 (ARGX-110) andwhich exhibit the same combination of binding characteristics, namely:

-   -   (a) binding within the amino acid sequence HIQVTLAICSS (SEQ ID        NO:342) in human CD70;    -   (b) cross-reactivity with CD70 homologs of rhesus macaque        (Macaca mulatta) and cynomolgus monkey (Macaca cynomolgus);    -   (c) binding to native and heat denatured recombinant human CD70.        Polynucleotides Encoding CD70 Antibodies

The invention also provides polynucleotide molecules encoding the CD70antibodies of the invention, also expression vectors containing anucleotide sequences which encode the CD70 antibodies of the inventionoperably linked to regulatory sequences which permit expression of theantigen binding polypeptide in a host cell or cell-free expressionsystem, and a host cell or cell-free expression system containing thisexpression vector.

Polynucleotide molecules encoding the CD70 antibodies of the inventioninclude, for example, recombinant DNA molecules. The terms “nucleicacid”, “polynucleotide” or a “polynucleotide molecule” as used hereininterchangeably and refer to any DNA or RNA molecule, either single- ordouble-stranded and, if single-stranded, the molecule of itscomplementary sequence. In discussing nucleic acid molecules, a sequenceor structure of a particular nucleic acid molecule may be describedherein according to the normal convention of providing the sequence inthe 5′ to 3′ direction. In some embodiments of the invention, nucleicacids or polynucleotides are “isolated.” This term, when applied to anucleic acid molecule, refers to a nucleic acid molecule that isseparated from sequences with which it is immediately contiguous in thenaturally occurring genome of the organism in which it originated. Forexample, an “isolated nucleic acid” may comprise a DNA molecule insertedinto a vector, such as a plasmid or virus vector, or integrated into thegenomic DNA of a prokaryotic or eukaryotic cell or non-human hostorganism. When applied to RNA, the term “isolated polynucleotide” refersprimarily to an RNA molecule encoded by an isolated DNA molecule asdefined above. Alternatively, the term may refer to an RNA molecule thathas been purified/separated from other nucleic acids with which it wouldbe associated in its natural state (i.e., in cells or tissues). Anisolated polynucleotide (either DNA or RNA) may further represent amolecule produced directly by biological or synthetic means andseparated from other components present during its production.

In one embodiment, the invention provides nucleotide sequences whichencode the VH domain and VL domain of the germlined llama-derived Fabdenoted herein 41D12, wherein the VH domain has the amino acid sequenceshown as SEQ ID NO:223 and the VH domain has the amino acid sequenceshown as SEQ ID NO:241. In a preferred embodiment the nucleotidesequence encoding the VH domain having the amino acid sequence shown asSEQ ID NO:223 comprises the nucleotide sequence shown as SEQ ID NO:344,and the nucleotide sequence encoding the VL domain having the amino acidsequence shown as SEQ ID NO:241 comprises the nucleotide sequence shownas SEQ ID NO:345.

In one embodiment, the invention provides nucleotide sequences whichencode the VH domain and VL domain of the germlined llama-derived Fabdenoted herein 57B6, wherein the VH domain has the amino acid sequenceshown as SEQ ID NO:274 and the VH domain has the amino acid sequenceshown as SEQ ID NO:276. In a preferred embodiment the nucleotidesequence encoding the VH domain having the amino acid sequence shown asSEQ ID NO:274 comprises the nucleotide sequence shown as SEQ ID NO:346,and the nucleotide sequence encoding the VL domain having the amino acidsequence shown as SEQ ID NO:276 comprises the nucleotide sequence shownas SEQ ID NO:347.

In one embodiment, the invention provides nucleotide sequences whichencode the VH domain and VL domain of the germlined llama-derived Fabdenoted herein 59D10, wherein the VH domain has the amino acid sequenceshown as SEQ ID NO:275 and the VH domain has the amino acid sequenceshown as SEQ ID NO:277. In a preferred embodiment the nucleotidesequence encoding the VH domain having the amino acid sequence shown asSEQ ID NO:275 comprises the nucleotide sequence shown as SEQ ID NO:348,and the nucleotide sequence encoding the VL domain having the amino acidsequence shown as SEQ ID NO:277 comprises the nucleotide sequence shownas SEQ ID NO:349.

For recombinant production of a CD70 antibody according to theinvention, a recombinant polynucleotide encoding it may be prepared(using standard molecular biology techniques) and inserted into areplicable vector for expression in a chosen host cell, or a cell-freeexpression system. Suitable host cells may be prokaryote, yeast, orhigher eukaryote cells, specifically mammalian cells. Examples of usefulmammalian host cell lines are monkey kidney CV1 line transformed by SV40(COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cellssubcloned for growth in suspension culture, Graham et al., J. Gen.Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10);Chinese hamster ovary cells/−DHFR(CHO, Urlaub et al., Proc. Natl. Acad.Sci. USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol.Reprod. 23:243-251 (1980)); mouse myeloma cells SP2/0-AG14 (ATCC CRL1581; ATCC CRL 8287) or NS0 (HPA culture collections no. 85110503);monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells(VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells(BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); humanliver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCCCCL51); TR1 cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68(1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2), aswell as DSM's PERC-6 cell line. Expression vectors suitable for use ineach of these host cells are also generally known in the art.

It should be noted that the term “host cell” generally refers to acultured cell line. Whole human beings into which an expression vectorencoding an antigen binding polypeptide according to the invention hasbeen introduced are explicitly excluded from the definition of a “hostcell”.

Antibody Production

In an important aspect, the invention also provides a method ofproducing a CD70 antibody of the invention which comprises culturing ahost cell (or cell free expression system) containing polynucleotide(e.g. an expression vector) encoding the CD70 antibody under conditionswhich permit expression of the CD70 antibody, and recovering theexpressed CD70 antibody. This recombinant expression process can be usedfor large scale production of CD70 antibodies according to theinvention, including monoclonal antibodies intended for humantherapeutic use. Suitable vectors, cell lines and production processesfor large scale manufacture of recombinant antibodies suitable for invivo therapeutic use are generally available in the art and will be wellknown to the skilled person.

As noted elsewhere, the CD70 antibodies provided herein, including inparticular antibodies based on the Fab 27B3 and its germlined variantssuch as 41D12 (ARGX-110) display characteristics which are particularlybeneficial for large-scale commercial manufacture. Specifically, theextremely high production yield (>4 g/L) achievable in a commercialscale recombinant expression system will dramatically reduce productioncosts.

Preferred embodiments also exhibit thermal stability when tested at 37°C. and also over several freeze-thaw cycles, which is again extremelybeneficial for commercial manufacture and storage of a clinical product.Surprisingly, several of the human germlined variants of Fab 27B3,including 41D12 and 40F1, exhibit improved thermal stability compared to27B3 itself.

Accordingly, in a further aspect the invention provides an isolatedantibody or antigen binding fragment thereof which binds to human CD70,said antibody or fragment comprising a heavy chain variable domaincorresponding to the amino acid sequence set forth in SEQ ID NO:178,provided that at least one amino acid at a Kabat position selected fromthe group consisting of H6, H18, H24, H31, H56, H74, H74, H77, H79, H83,H84, H89, H93, H94, H108, H110, and H112 is substituted with anotheramino acid.

This antibody, or antigen binding fragment thereof, may exhibit greaterthermal stability than an antibody, or antigen binding fragment thereof,comprising a heavy chain variable domain with the amino acid sequenceset forth in SEQ ID NO:178.

In a further embodiment there is provided a CD70 antibody or antigenbinding fragment which comprises a light chain variable domaincorresponding to the amino acid sequence set forth in SEQ ID NO:201,provided that at least one amino acid at a Kabat position selected fromthe group consisting of L2, L11, L12, L25, L26, L30, L46, L53, L60, L61,L67, L68, L76, L80, L81, L85, and L87 is substituted with another aminoacid.

This antibody, or antigen binding fragment thereof, may exhibit greaterthermal stability than an antibody, or antigen binding fragment thereof,comprising a heavy chain variable domain with the amino acid sequenceset forth in SEQ ID NO:201.

In a particular embodiment, the CD70 antibody, or antigen bindingfragment thereof may comprise:

a) a heavy chain variable domain with the amino acid sequence set forthin SEQ ID NO:178 comprising one or more amino acid substitutions atKabat positions selected from the group consisting of H6, H18, H24, H31,H56, H74, H74, H77, H79, H83, H84, H89, H93, H94, H108, H110, and H112;andb) a light chain variable domain with the amino acid sequence set forthin SEQ ID NO:201 comprising one or more amino acid substitutions atKabat positions selected from the group consisting of L2, L11, L12, L25,L26, L30, L46, L53, L60, L61, L67, L68, L76, L80, L81, L85, and L87.

This antibody, or antigen binding fragment thereof, of claim 24, mayexhibit greater thermal stability than an antibody, or antigen bindingfragment thereof, comprising a heavy chain variable domain with theamino acid sequence set forth in SEQ ID NO:178 and a light chainvariable domain with the amino acid sequence set forth in SEQ ID NO:201.

Therapeutic Utility of CD70 Antibodies

The CD70 antibodies, or antigen binding fragments thereof, providedherein can be used to inhibit the growth of cancerous tumour cells invivo and are therefore useful in the treatment of CD70-expressingcancers.

Accordingly, further aspects of the invention relate to methods ofinhibiting tumour cell growth in a human patient, and also methods oftreating or preventing cancer, which comprise administering to a patientin need thereof a therapeutically effective amount of a CD70 antibody orantigen binding fragment as described herein.

Another aspect of the invention provides a CD70 antibody or antigenbinding fragment as described herein for use inhibiting the growth ofCD70-expressing tumour cells in a human patient.

A still further aspect of the invention provides a CD70 antibody orantigen binding fragment as described herein for use treating orpreventing cancer in a human patient.

Preferred cancers whose growth may be inhibited using the CD70antibodies described herein include renal cancer (e.g., renal cellcarcinoma), breast cancer, brain tumors, chronic or acute leukemiasincluding acute myeloid leukemia, chronic myeloid leukemia, acutelymphoblastic leukemia, chronic lymphocytic leukemia, lymphomas (e.g.,Hodgkin's and non-Hodgkin's lymphoma, lymphocytic lymphoma, primary CNSlymphoma, T-cell lymphoma), nasopharyngeal carcinomas, melanoma {e.g.,metastatic malignant melanoma), prostate cancer, colon cancer, lungcancer, bone cancer, pancreatic cancer, skin cancer, cancer of the heador neck, cutaneous or intraocular malignant melanoma, uterine cancer,ovarian cancer, rectal cancer, cancer of the anal region, stomachcancer, testicular cancer, uterine cancer, carcinoma of the fallopiantubes, carcinoma of the endometrium, carcinoma of the cervix, carcinomaof the vagina, carcinoma of the vulva, cancer of the esophagus, cancerof the small intestine, cancer of the endocrine system, cancer of thethyroid gland, cancer of the parathyroid gland, cancer of the adrenalgland, sarcoma of soft tissue, cancer of the urethra, cancer of thepenis, solid tumors of childhood, cancer of the bladder, cancer of thekidney or ureter, carcinoma of the renal pelvis, neoplasm of the centralnervous system (CNS), tumor angiogenesis, spinal axis tumor, brain stemglioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamouscell cancer, mesothelioma.

The CD70 antibodies described herein can also be used to treat a subjectwith a tumorigenic disorder characterized by the presence of tumor cellsexpressing CD70 including, for example, renal cell carcinomas (RCC),such as clear cell RCC, glioblastoma, breast cancer, brain tumors,nasopharangeal carcinomas, non-Hodgkin's lymphoma (NHL), acutelymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL),Burkitt's lymphoma, anaplastic large-cell lymphomas (ALCL), multiplemyeloma, cutaneous T-cell lymphomas, nodular small cleaved-celllymphomas, lymphocytic lymphomas, peripheral T-cell lymphomas, Lennert'slymphomas, immunoblastic lymphomas, T-cell leukemia/lymphomas (ATLL),adult T-cell leukemia (T-ALL), entroblastic/centrocytic (cb/cc)follicular lymphomas cancers, diffuse large cell lymphomas of B lineage,angioimmunoblastic lymphadenopathy (AILD)-like T cell lymphoma, HIVassociated body cavity based lymphomas, embryonal carcinomas,undifferentiated carcinomas of the rhino-pharynx (e.g., Schmincke'stumor), Castleman's disease, Kaposi's Sarcoma, multiple myeloma,Waldenstrom's macroglobulinemia, Mantle cell lymphoma and other B-celllymphomas.

Specific embodiments relate to treatment of any one of the above-listedcancers with an antibody comprising the llama-derived Fab 27B3, or oneof its germlined variants, including in particular 41D12 (ARGX-110). Inparticular, any one of the above-listed cancers may be treated using anantibody comprising the Fab regions of 41D12 (germlined variant of 27B3)fused to the constant regions of human IgG1. In the latter embodiment,the human IgG1 constant region may be further engineered in order tomaximise antibody effector functions (e.g. by addition of pointmutations), or the 41D12-IgG1 antibody may be non-fucosylated, again toenhance antibody effector function.

As noted elsewhere, the CD70 antibodies described herein, particularly27B3 and its germlined variants, exhibit particularly strong binding tohuman cancer cell lines which express cell-surface CD70 at low copynumber, such as for example the large B cell lymphoma cell lineSU-DHL-6, the chronic lymphocytic leukemia cell line JVM-2, thecutaneous T cell lymphoma cell line HH, the gastric carcinoma cell lineMKN-45, the lung carcinoma cell lines A549 and EBC-1, the melanoma celllines WM852 and WM793, the glioblastoma cell line GaMG and the ovariancarcinoma cell lines OAW-42, SKOv3 and OVCAR3. Indeed, the affinity of41D12 (ARGX-110) for these low copy-number cancer cell lines issignificantly higher than comparator prior art antibodies, such as SGN70and MDX1411.

The “improved” affinity for low copy-number cancer cell lines is ofdirect relevance for clinical treatment of the corresponding cancers, asdemonstrated by the cell spiking experiments in the accompanyingexamples. When cells of a low copy-number cell line (e.g. the diffuselarge B cell lymphoma cell line SU-DHL-6) are spiked into freshlyisolated PBMCs from healthy donors, cells of the cancer cell line arepreferentially depleted by the example CD70 antibody ARGX-110.

Accordingly, a further aspect of the invention relates to a method oftreating or preventing cancer, which comprises administering to apatient in need thereof a therapeutically effective amount of a CD70antibody or antigen binding fragment as described herein, wherein thecancer is a cancer which exhibits low copy-number expression of CD70.

The invention also provides a CD70 antibody or antigen binding fragmentas described herein for use treating or preventing cancer in a humanpatient, wherein the cancer is a cancer which exhibits low copy-numberexpression of CD70.

In each of these aspects the low copy-number cancer is preferablyselected from the group consisting of: large B cell lymphoma, chroniclymphocytic leukaemia, cutaneous T cell lymphoma, gastric cancer, lungcancer, melanoma, glioblastoma and ovarian cancer.

Specific embodiments relate to treatment of low copy-number cancers withan antibody comprising the llama-derived Fab 27B3, or one of itsgermlined variants, including in particular 41D12 (ARGX-110). Inparticular, any one of the above-listed low copy-number cancers may betreated using an antibody comprising the Fab regions of 41D12 (germlinedvariant of 27B3) fused to the constant regions of human IgG1.

The CD70 antibodies described herein are also useful for the treatmentof immunological disorders characterised by expression of CD70. Specificexamples of such immunological disorders include the following:rheumatoid arthritis, autoimmune demyelinative diseases (e.g., multiplesclerosis, allergic encephalomyelitis), endocrine ophthalmopathy,uveoretinitis, systemic lupus erythematosus, myasthenia gravis, Grave'sdisease, glomerulonephritis, autoimmune hepatological disorder,inflammatory bowel disease (e.g., Crohn's disease, ulcerative colitis,Celiac disease), anaphylaxis, allergic reaction, Sjogren's syndrome,type I diabetes mellitus, primary biliary cirrhosis, Wegener'sgranulomatosis, fibromyalgia, polymyositis, dermatomyositis, multipleendocrine failure, Schmidt's syndrome, autoimmune uveitis, Addison'sdisease, adrenalitis, thyroiditis, Hashimoto's thyroiditis, autoimmunethyroid disease, pernicious anemia, gastric atrophy, chronic hepatitis,lupoid hepatitis, atherosclerosis, subacute cutaneous lupuserythematosus, hypoparathyroidism, Dressier's syndrome, autoimmunethrombocytopenia, idiopathic thrombocytopenic purpura, hemolytic anemia,pemphigus vulgaris, pemphigus, dermatitis herpetiformis, alopeciagreata, pemphigoid, scleroderma, progressive systemic sclerosis, CRESTsyndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility,sclerodactyl), and telangiectasia), male and female autoimmuneinfertility, ankylosing spondolytis, ulcerative colitis, mixedconnective tissue disease, polyarteritis nedosa, systemic necrotizingvasculitis, atopic dermatitis, atopic rhinitis, Goodpasture's syndrome,Chagas' disease, sarcoidosis, rheumatic fever, asthma, recurrentabortion, anti-phospholipid syndrome, farmer's lung, erythemamultiforme, post cardiotomy syndrome, Gushing's syndrome, autoimmunechronic active hepatitis, bird-fancier's lung, toxic epidermalnecrolysis, Alport's syndrome, alveolitis, allergic alveolitis,fibrosing alveolitis, interstitial lung disease, erythema nodosum,pyoderma gangrenosum, transfusion reaction, Takayasu's arteritis,polymyalgia rheumatica, temporal arteritis, schistosomiasis, giant cellarteritis, ascariasis, aspergillosis, Sampter's syndrome, eczema,lymphomatoid granulomatosis, Behcet's disease, Caplan's syndrome,Kawasaki's disease, dengue, encephalomyelitis, endocarditis,endomyocardial fibrosis, endophthalmitis, erythema elevatum et diutinum,psoriasis, erythroblastosis fetalis, eosinophilic faciitis, Shulman'ssyndrome, Felty's syndrome, filariasis, cyclitis, chronic cyclitis,heterochronic cyclitis, Fuch's cyclitis, IgA nephropathy,Henoch-Schonlein purpura, graft versus host disease, transplantationrejection, cardiomyopathy, Eaton-Lambert syndrome, relapsingpolychondritis, cryoglobulinemia, Evan's syndrome, and autoimmunegonadal failure, disorders of B lymphocytes (e.g., systemic lupuserythematosus, Goodpasture's syndrome, rheumatoid arthritis, and type Idiabetes), Th1-lymphocytes (e.g., rheumatoid arthritis, multiplesclerosis, psoriasis, Sjorgren's syndrome, Hashimoto's thyroiditis,Grave's disease, primary biliary cirrhosis, Wegener's granulomatosis,tuberculosis, or graft versus host disease), or Th2-lymphocytes (e.g.,atopic dermatitis, systemic lupus erythematosus, atopic asthma,rhinoconjunctivitis, allergic rhinitis, Omenn's syndrome, systemicsclerosis, or chronic graft versus host disease), Churg Strausssyndrome, microscopic polyangiitis and Takayasu's arteritis.

Specific embodiments relate to treatment of any one of the above-listedimmunological disorders with an antibody comprising the llama-derivedFab 27B3, or one of its germlined variants, including in particular41D12 (ARGX-110). In particular, any one of the above-listed cancers maybe treated using an antibody comprising the Fab regions of 41D12(germlined variant of 27B3) fused to the constant regions of human IgG1.In the latter embodiment, the human IgG1 constant region may be furtherengineered in order to maximise antibody effector functions (e.g. byaddition of point mutations), or the 41D12-IgG1 antibody may benon-fucosylated, again to enhance antibody effector function.

As used herein, the term “treating” or “treatment” means slowing,interrupting, arresting, controlling, ameliorating, stopping, reducingseverity of a symptom, disorder, condition or disease, but does notnecessarily involve a total elimination of all disease-related symptoms,conditions or disorders.

For human therapeutic use the CD70 antibodies described herein may beadministered to a human subject in need of treatment in an “effectiveamount”. The term “effective amount” refers to the amount or dose of aCD70 antibody which, upon single or multiple dose administration to ahuman patient, provides therapeutic efficacy in the treatment ofdisease. Therapeutically effective amounts of the CD70 antibody cancomprise an amount in the range of from about 0.1 mg/kg to about 20mg/kg per single dose. A therapeutic effective amount for any individualpatient can be determined by the healthcare professional by monitoringthe effect of the CD70 antibody on a biomarker, such as cell surfaceCD70 in tumour tissues, or a symptom such as tumour regression, etc. Theamount of antibody administered at any given time point may be varied sothat optimal amounts of CD70 antibody, whether employed alone or incombination with any other therapeutic agent, are administered duringthe course of treatment.

It is also contemplated to administer the CD70 antibodies describedherein, or pharmaceutical compositions comprising such antibodies, incombination with any other cancer treatment, as a combination therapy.

Use of Antibodies which are Poorly Internalised to DepleteCD70-Expressing Cells

A surprising finding described herein is that CD70 antibodies bound toCD70 expressed on the surface of cancer cell lines are poorlyinternalised. This is a surprising result, since the prior art haspreviously described CD70 as an “internalising target”. Although thereis variation between different cancer cell types in the precise degreeof internalisation of bound CD70 antibodies, no cancer cell type isobserved to “fully internalise” the bound CD70 antibody. For example,renal cancer cell lines previously described as rapidly internalising infact are shown to internalise a maximum of about 70% of bound antibodyARGX-110. Many other cancer cell lines internalise less than 30%, lessthan 20% or even less than 10% of bound ARGX-110.

The variation in degree of internalisation between different cancer celllines is largely disease-indication dependent, with similar resultsbeing observed using different CD70 antibodies. However, the poorinternalisation in certain cell lines is particularly apparent whenusing the antibody ARGX-110. The observed low degree of internalisationon many cancer cells provides a rationale for the use of CD70 antibodieswith strong effector function in order to deplete (e g kill or inhibitthe growth of) cancer cells in vivo. Since bound CD70 antibodies arerelatively poorly internalised on many cancer cells, the antibody willremain bound to the cell surface and can thereby trigger effectorfunctions (ADCC, CDC, ADCP) by interactions with the immune system of ahuman patient.

Accordingly, a further important aspect of the invention relates to amethod of depleting CD70-expressing cells in a human patient, comprisingadministering to said patient an effective amount of an antibody whichbinds to human CD70, wherein said CD70-expressing cells exhibit 30% orless, 25% or less, 20% or less, 15% or less, 10% or less or 5% or lessinternalisation of said antibody after a period of 6 hours, and whereinsaid antibody exhibits at least one effector function selected from thegroup consisting of ADCC, CDC and ADCP.

In this method, the CD70 antibody (with active effector functions) isused to deplete CD70 expressing cells. This depletion of CD70-expressingcells may form the basis of therapeutic or prophylactic treatment, e.g.for treatment of prevention of CD70-expressing cancers, or for treatmentof prevention of CD70-associated immunological disorders. The precisemechanism by which CD70-expressing antibodies are depleted may depend onthe type of effector functions exhibited by the CD70 antibody, but willbe dependent on strong (high affinity) binding of the CD70 antibody tocell-surface CD70, coupled with poor internalisation of the boundantibody.

In certain embodiments the CD70-expressing cells are CD70-expressingcancer cells. In particular, the CD70-expressing cancer cells may be ofa cancer type selected from the group consisting of: Burkitt lymphoma,large B cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, mantlecell lymphoma, chronic lymphocytic leukemia, pancreatic carcinoma,gastric carcinoma, glioblastoma and lung carcinoma. Cell lines derivedfrom each of these cancers have been demonstrated to internalise 30% orless of bound CD70 antibody over a period of 6 hours. In a furtherembodiment CD70-expressing cancer cells may be of a cancer type selectedfrom the group consisting of: Burkitt lymphoma, large B cell lymphoma,Hodgkin lymphoma, non-Hodgkin lymphoma, mantle cell lymphoma, chroniclymphocytic leukemia, gastric carcinoma, glioblastoma and lungcarcinoma. Cell lines derived from each of these cancers have beendemonstrated to internalise 20% or less of bound CD70 antibody over aperiod of 6 hours. In a further embodiment CD70-expressing cancer cellsmay be of a cancer type selected from the group consisting of: Burkittlymphoma, large B cell lymphoma, mantle cell lymphoma, chroniclymphocytic leukemia, gastric carcinoma and lung carcinoma. Cell linesderived from each of these cancers have been demonstrated to internalise10% or less of bound CD70 antibody over a period of 6 hours.

In other embodiments the CD70-expressing cells may be CD70-expressingactivated T cells, which do not exhibit any detectable internalisationof bound CD70 antibodies (specifically no internalisation of ARGX-110 isobserved over a period of 6 hours. This observation that bound CD70antibodies are not internalised by CD70+ activated T cells is stronglysupportive of the use of CD70 antibodies with strong effector function(ADCC, CDC or ADCP), including but not limited to ARGX-110, in thetreatment of immunological diseases mediated by CD70+ activated T cells,including autoimmune disorders such as for example rheumatoid arthritis,psoriasis, SLE, etc.

The above-listed embodiments can utilise essentially any CD70 antibody.In specific embodiments the antibody can be any CD70 antibody which hasbeen shown experimentally to exhibit 30% or less, 25% or less, 20% orless, 15% or less, 10% or less or 5% or less internalisation after aperiod of 6 hours in a CD70-expressing cancer cell line selected fromthe group consisting of Raji, SU-DHL-6, MHHPREB1, Mino, Mec1, JVM-2,MKN-45, A549, U87MG, PANC-1, PANC-89, L428, Granta 519, Rec-1 and EBC-1.In effect, the cell line internalisation assay described in theaccompanying examples can be used as a screen both to determine whetherparticular disease indications should be targeted with CD70 antibodieshaving strong effector function, or with CD antibody-drug conjugates(discussed below), and also to identify suitable CD70 antibodies for usein treating particular disease indications.

Accordingly, the invention also provides a method of depletingCD70-expressing cancer cells in a human patient, comprisingadministering to said patient an effective amount of an antibody whichbinds to human CD70, wherein said antibody exhibits 30% or lessinternalisation after a period of 6 hours in a CD70-expressing cancercell line selected from the group consisting of Raji, SU-DHL-6,MHHPREB1, Mino, Mec1, JVM-2, MKN-45, A549, U87MG, PANC-1, PANC-89, L428,Granta 519, Rec-1 and EBC-1, and wherein said antibody exhibits at leastone effector function selected from the group consisting of ADCC, CDCand ADCP. In one embodiment the antibody to be used to depleteCD70-expressing cancer cells in a human patient may be an antibody whichexhibits 10% or less internalisation over a period of 6 hours in aCD70-expressing cancer cell line selected from the group consisting ofRaji, SU-DHL-6, Granta 519, Rec-1, Mec1, JVM-2, MKN-45, A549 and EBC-1,and also exhibits at least one effector function selected from the groupconsisting of ADCC, CDC and ADCP.

Specific embodiments of this aspect of the invention may utilise any ofthe CD70 antibodies described herein, which bind human CD70 withextremely high affinity. Preferred embodiments may utilise an antibodycomprising the llama-derived Fab 27B3, or one of its germlined variants,including in particular 41D12 (ARGX-110). Particularly preferredembodiments utilise an antibody comprising the Fab regions of 41D12(germlined variant of 27B3) fused to the constant regions of human IgG1.In the latter embodiment, the human IgG1 constant region may be furtherengineered in order to maximise antibody effector functions (e.g. byaddition of point mutations), or the 41D12-IgG1 antibody may benon-fucosylated, again to enhance antibody effector function.

Since the rationale behind the use of poorly internalising CD70antibodies to deplete CD70-expressing cells relies on antibody effectorfunctions, it is preferred that the antibodies used in this aspect ofthe invention are not immunoconjugates in which the CD70 antibody islinked to a pharmaceutical agent, cytotoxic agent, cytostatic agent,drug moiety, etc. Indeed, a particular advantage of this aspect of theinvention is avoidance of the need to use antibody-drug conjugates,which is desirable both from a patient-safety perspective (avoidsadministration of potentially toxic agents) and an economic perspective(avoids costly synthesis of antibody-drug conjugates).

Use of Antibody-Drug Conjugates to Kill Cells with SignificantInternalisation CD70 Antibodies

It is demonstrated in the present examples that there are significantcell-to-cell differences in the degree of internalisation of CD70antibodies, and that these differences are largely dependent on celltype, rather than the nature of the CD70 antibody (although differencesare particularly marked when using the antibody ARGX-110). For thosecell types which exhibit a significant degree of internalisation ofbound CD70 antibody, i.e. 30% or more, preferably 40% or more, morepreferably 50% or more, or even 60% or more internalisation after 6hours, an alternative therapeutic strategy may be based on the use ofCD70 antibody-drug conjugates.

Accordingly, a further important aspect of the invention relates to amethod of depleting CD70-expressing cells in a human patient, comprisingadministering to said patient an effective amount of an antibody-drugconjugate comprising an antibody which binds to human CD70 and acytotoxic or cytostatic drug moiety, wherein said CD70-expressing cellsexhibit 30% or more, 40% or more, 50% or more, or 60% or moreinternalisation of said antibody which binds to human CD70 after aperiod of 6 hours.

In specific embodiments the CD70-expressing cells may internalisebetween 40% and 70%, preferably between 50% and 70% of the CD70 antibodywhich forms the basis of the antibody-drug conjugate within a period of6 hours.

In this method, the CD70 antibody-drug conjugate is used to deplete CD70expressing cells. This depletion of CD70-expressing cells may form thebasis of therapeutic or prophylactic treatment, e.g. for treatment ofprevention of CD70-expressing cancers, or for treatment of prevention ofCD70-associated immunological disorders. The precise mechanism by whichCD70-expressing antibodies are depleted may depend on the nature of theantibody-drug conjugate, i.e. whether the “drug” moiety is cytotoxic orcytostatic, but will be dependent on strong (high affinity) binding ofthe CD70 antibody to cell-surface CD70, coupled with significantinternalisation of the bound antibody.

In certain embodiments the CD70-expressing cells are CD70-expressingcancer cells. In particular, the CD70-expressing cancer cells may be ofa cancer type selected from the group consisting of: cutaneous T celllymphoma, multiple myeloma, renal cell carcinoma, astrocytoma, melanomaand ovarian carcinoma. Cell lines derived from each of these cancershave been demonstrated to internalise more than 30% of bound CD70antibody over a period of 6 hours. In a further embodiment, theCD70-expressing cancer cells may be of a cancer type selected from thegroup consisting of: renal cell carcinoma, astrocytoma, melanoma andovarian carcinoma. Cell lines derived from each of these cancers havebeen demonstrated to internalise more than 50% of bound CD70 antibodyover a period of 6 hours.

In specific embodiments of this aspect of the invention, theantibody-drug conjugate may be based on any of the CD70 antibodiesdescribed herein, which bind human CD70 with extremely high affinity. Inpreferred embodiments the antibody-drug conjugate may be based on anantibody comprising the llama-derived Fab 27B3, or one of its germlinedvariants, including in particular 41D12. The features of antibody-drugconjugates intended for human therapeutic use are generally known in theart, particularly with regard to the nature of the “drug” moiety, e.g. acytotoxic or cytostatic agent, and means of conjugation of the drugmoiety to the CD70 antibody moiety. Examples of CD70 antibody-drugconjugates are given, for example, in WO 2004/073656.

Pharmaceutical Compositions

The scope of the invention includes pharmaceutical compositions,containing one or a combination of CD70 antibodies of the invention, orantigen-binding fragments thereof, formulated with one or more apharmaceutically acceptable carriers or excipients. Such compositionsmay include one or a combination of (e.g., two or more different) CD70antibodies.

Techniques for formulating antibodies for human therapeutic use are wellknown in the art and are reviewed, for example, in Wang et al., Journalof Pharmaceutical Sciences, Vol. 96, pp 1-26, 2007.

INCORPORATION BY REFERENCE

Various publications are cited in the foregoing description andthroughout the following examples, each of which is incorporated byreference herein in its entirety.

EXAMPLES

The invention will be further understood with reference to the followingnon-limiting experimental examples.

Example 1: Immunization of Llamas

Immunizations of llamas and harvesting of peripheral blood lymphocytes(PBLs) as well as the subsequent extraction of RNA and amplification ofantibody fragments were performed as described by De Haard andcolleagues (De Haard H, et al., J. Bact. 187:4531-4541, 2005). Twollamas were immunized with CD70-expressing 786-O cells (ATCC-CRL-1932)and two llamas with CD70-expressing Raji cells (ATCC-CCL-86). Cells wereprepared freshly for each immunization and were verified for CD70expression by FACS analysis. The llamas were immunized withapproximately 10⁷ live cells injected intramuscularly in the neck, onceper week for six weeks. Freund's Incomplete Adjuvant was injected in theneck muscles a few centimeters away from the site of cellularimmunization.

Blood samples of 10 ml were collected pre- and post immunization toinvestigate the immune response. The sera from the llamas were testedfor the presence of antibodies against recombinant CD70 (Flag-TNC-CD70)by ELISA prior to (day 0) and after (day 55 or 69) immunization. Itshould be noted that as detection antibody the goat anti-llama IgG1/2(Bethyl, A160-100P) was used that does not discriminate betweenconventional and heavy chain antibodies, meaning that the measured CD70response is from the total IgG. The results are shown in FIG. 1.

Three-to-four days after the last immunization, 400 ml blood wascollected for extraction of total RNA from the PBLs using a Ficoll-Paquegradient to isolate PBLs and the method described by Chomczynski P, etal., Anal. Biochem. 162: 156-159, 1987 to prepare the RNA. In average,RNA yields of 450 μg were achieved, of which an 80 μg aliquot was usedfor random cDNA synthesis and subsequent amplification of VHCH1, VλCλand VκCκ gene segments.

Example 2: Library Construction

Independent VλCλ and VκCκ libraries were constructed using a two stepPCR, in which 25 cycles with non tagged primers was done followed by 10cycles using tagged version of these primers (De Haard H, et al., Biol.Chem. 274, 1999). The VHCH1 libraries were built in parallel using thesame approach. The sizes of the individual libraries were between 10⁷and 10¹⁰ cfu. Next, the light chain from the VλCλ and VκCκ librarieswere re-cloned separately in the VHCH1-expressing vector to create the“Lambda” and “Kappa” llama Fab-library respectively. Alternatively thedigested VHCH1 amplicons were directly cloned into the VκCκ and VλCλlibraries avoiding the construction of a separate VHCH1 library. Aslight chain shuffling generally delivers better affinity variants thanheavy chain shuffling does, we chose to generate precloned light chainlibraries and directly clone the heavy chain amplicons in these primaryrepertoires.

The final phage display libraries were between 5×10⁸ and 2×10⁹ cfu.Quality control of the libraries by analysis of percentage of fulllength Fab containing clones was routinely performed using PCR.

Example 3: Selections and Screening

Two-to-three rounds of phage selections were done on either capturedrecombinant CD70 (Flag-TNC-CD70) or on directly coated CD70 usingstandard protocols. Elution of bound phage was done with trypsin.

From selected phage transfected into TG1, individual colonies werepicked randomly for growth in 96 well plates. After IPTG inductionaccording to standard protocols periplasmic fractions (penis) containingFabs were prepared. In all selections and for all four llamas, severalclones were found that were able to express Fabs capable in inhibitingthe interaction between CD27 and CD70 as determined by the inhibitionELISA (FIGS. 2 and 3). In this assay, Flag-TNC-CD70 is captured by ananti-Flag mAb and binding of CD27-Fc is detected via a biotinylatedanti-CD27 mAb and strep-HRP in the absence or presence of CD70 Fabs(FIGS. 2 and 3) or CD70 mAbs (FIG. 4). In FIG. 2B it can be seen that27B3, which has an identical VH as 9D1, but contains a somewhatdifferent light chain, seems to block binding of CD27 to CD70 moreefficiently, which was in line with the better off rate of this Fab asmeasured on Biacore (data not shown). Table 3 indicates the blockingpotency (expressed as IC50) for the various llama-human chimeric mAbstested in the CD70 inhibition ELISA. Fab 27B3 has an IC50 of around 0.4nM, which even seems to improve upon formatting into a chimericllama-human IgG1 yielding an IC 50 of 0.25 nM, whereas mAb 9D1 with theother light chain is 3-fold less potent (0.73 nM). FIG. 3 is a compositeof an experiment testing llama CD70 Fabs for blocking activity (in theinhibition ELISA, see white bars) and for CD70 binding (binding ELISA,black bars). For the binding ELISA, Flag-TNC-CD70 is captured by ananti-Flag mAb and binding of the Fabs is detected using an anti-myc-HRPmAb, which recognizes the MYC tag fused to the C-terminus of the Fdfragment. For comparison purposes, known monoclonal antibodies to CD70(SGN70 described in US 2010/0129362 A1, clone 1F6; and MDX69A7 describedin WO2008/074004 FIGS. 5A and 5B) were tested in parallel. Manyllama-derived Fab clones were identified that demonstrate pure bindingin ELISA and are non-blocking (see FIG. 3—for example Fab clones 45H8,45C4, 46A2, 46G7, 46E4, 46F12 and 46G12). Of interest is clone 59D10,which as Fab has no blocking activity, but when converted into achimeric IgG was antagonistic (see Table 3). Amino acid sequences of theVH and VL domains of binding and blocking llama-derived Fab clones areshown elsewhere herein (Tables 6-9).

TABLE 3 Blocking potency expressed as IC50 (ng/ml) for individual SIMPLEantibodies (mAbs) and benchmarks SGN70 and MDX69A7 as measured in theCD70 inhibition ELISA using recombinant human CD70. mAb IC50 (ng/ml)SGN70 330 MDX69A7 4038  9D1 109  5F4 296  9E1 1400  9G2 40  5B2 92  9B2126  4D2 254 24D4 84 24B6 420 27B3 38 19G10 75 57B6 154 59D10 107mAbs 27B3 and 57B6 was also tested for their ability to inhibit theinteraction between rhesus recombinant CD70 (FLAG-TNC-rhesus CD70) andCD27. The IC50 value determined by the inhibition ELISA described abovewas 150 and 135 ng/ml, respectively.

Example 4: Affinity for Human and Rhesus CD70

Off-rates of the purified llama-derived anti-CD70 blocking Fabs weredetermined using the Biacore. Recombinant human CD70 was immobilized ona CM5 Biacore chip. The immobilization was performed in accordance witha method provided by Biacore and by using the NHS/EDC kit (Biacore AB):after activation of the chip, a solution of 50 μg/ml of recombinant CD70in 10 mM acetate buffer with pH of 5 was prepared and 1 μl of thissolution (50 ng) was injected resulting in a surface density ofapproximately 1000 RU.

Fabs for the CD70 mAbs ARGX-110, MDX2H5 and SGN70 were prepared bytrypsin digestion (SGN70 and MDX69A7 are described elsewhere herein,MDX2H5, also referred to as MDX1411, is described in WO2008/074004 FIGS.1A and 1B). Fabs, at a concentration of approximately 100-400 μg/ml,were diluted 6-fold in hepes-buffered saline (0.1M Hepes, 1.5M NaCl, 30mM EDTA, 0.5% v/v surfactant P20). They were injected (30 μl) and passedthrough the flow cells at a flow rate of 30 μl/min. After binding of theFab to CD70, the off-rate was monitored for a period of 10 minutes.After the dissociation, the flow cell surfaces were regenerated byinjecting 5 μl of 10 mM NaOH. Sometimes multiple injections of NaOH wereneeded to regenerate the surfaces depending on the affinity of the Fabs.Off-rate analysis was done by applying the BIAevaluation software.First, the sensogram of the blank runs were subtracted from thoseobtained with the coated flow cell. Then the off-rate was determined fora time range of 10 minutes using the Fit kinetics application and theK_(d) value was calculated. Moreover, the off-rate of CD27 for CD70 wasdetermined as well. The off rates are summarized in Table 4.

All llama-derived Fabs tested bind with apparent very low off-rates(i.e. high affinity) to human recombinant CD70 and to rhesus recombinantCD70 based on Biacore off rates in the range of 0.4-4.8×10⁻⁴ s⁻¹. Theoff-rates of the llama-derived Fabs for human CD70 are much better thanfor the reference mAb derived Fabs (7-30×10⁻⁴ s⁻¹). The llama-derivedFabs with the best off-rates were shown to have comparable off-rates tothat of CD27 for its interaction with CD70 (0.8×10⁻⁴ s⁻¹). It should benoted that the lower limit for measurement of off-rates using Biacore isaround 0.4×10⁻⁴ s⁻¹. Hence the llama-derived Fabs with off-rates fallingclose to this limit as assessed by Biacore may in fact exhibit even ahigher affinity for CD70.

TABLE 4 Off rates of llama-derived Fabs and reference Fabs for human orrhesus CD70 k_(off) (s⁻¹) × E⁻⁴ k_(off) (s⁻¹) × E⁻⁴ Fab tested humanCD70 rhesus CD70 SGN70-Fab 7.0 MDX2H5-Fab 17 No binding MDX69A7-Fab 30CD27 0.8  1C2 0.4 3.2  9D1 4.8 2.0  9G2 1.8 1.8  5F4 0.8 5.0  9E1 1.92.0  9B2 2.8 6.1  4D2 3.3 2.9 27B3 0.8 24E3 0.8 33D8 4.5 24F2 1.6 24B61.0 19G10 0.8  8B12 3.6 45B12 2.1 45D9 2.1 45F8 2.0 57B6 1.0 0.4 59D1013.9 No binding

Example 5: Binding of Chimeric Llama-Human CD70 mAbs to CD70 PositiveCells

The VH and VL of interesting llama-derived Fab clones were fused toconstant regions of human IgG1 and to human Cλ (all variable light chainregions are of lambda origin) and produced as bivalent chimericmonoclonal antibodies in the system described in patent application WO2009/145606. The chimeric llama-human mAbs were purified on Protein Afollowed by gel filtration.

786-O cells were incubated with a 1/5 dilution series of chimericllama-human CD70 inhibiting mAbs (20 μg/ml-0.25 ng/ml) and binding ofthe mAbs to the cells was detected using an anti-human Fc-FITC antibody(1/500 diluted conjugate AF006 from supplier Binding Site). Fluorescenceof 10,000 cells/condition was measured using a flow cytometer and themedian fluorescence was plotted. The results are shown in FIG. 5. Mostof the mAbs bind with high affinity to 786-O cells. SIMPLE antibody 1C2,which as Fab showed to have the best off rate and blocking potency, wasnot able to recognize cell surface expressed CD70 (data not shown) andtherefore was not studied furthermore. SIMPLE antibody 27B3 has the bestaffinity for cell bound receptor with an EC50 of 0.24 nM.

In a further experiment, NHL-derived MHH-PREB-1 cells (10⁵ cells) wereincubated with a concentration gradient of chimeric llama-human CD70inhibiting mAbs (20 μg/ml-0.25 ng/ml) and binding of the mAbs to thecells was detected using anti-human IgG-FITC antibody. The results areshown in FIG. 5B. The EC50 values for 57B6 and 59D10 were 83 and 74ng/ml, respectively.

Example 6: Inhibition in Co-Culture Experiments by Chimeric Llama-HumanCD70 mAbs

In order to determine the blocking potency in a cell based assay thesystem described by Wyzgol and colleagues was used (J Immunol (2009)183: 1851-1861). Fibrosarcoma cell line HT1080 transfected with humanCD27 secretes IL-8 upon ligation with trimeric recombinant CD70 andblocking of this interaction with a neutralizing mAb can be measured byreduced cytokine levels. A modified version of the assay was applied inwhich the B cell lymphoma Raji cell line served as source of CD70,although the molecule is expressed on the cell surface and therefore ismore relevant than soluble CD70.

Raji cells were cultured and checked for CD70 expression by FACSanalysis. Cells were then mixed in a 96-well tissue culture plate(50,000 cells/well) with the anti-CD70 mAbs and incubated at RT for 1hour. HT1080-CD27 cells were added (10,000 cells/well) and thoroughlymixed. Co-cultures were transferred to the incubator and grown overnightat 37° C. Supernatants were collected and analyzed for their IL-8content by ELISA. The results for the SIMPLE antibodies are shown inFIG. 6 and are compared to reference CD70 mAbs. IC50 values for thechimeric llama-human CD70 mAbs are between 3 and 517 ng/ml as comparedto 42 ng/ml for SGN70 and 143 ng/ml for MDX69A7 (see table 5). SIMPLEantibodies 27B3 (IC50 of 33 pM) and 9G2 (IC50 of 20 pM) are 10 to 20fold more potent than benchmark SGN70 (IC50 of 370 pM) that again is 3fold more potent than benchmark MDX69A7 (1 nM). It can therefore beconcluded that the chimeric llama-human mAbs are extremely potent inblocking the interaction between CD27 and CD70 and outperform thebenchmark antibodies in this highly relevant bioassay.

TABLE 5 Neutralizing potency expressed as IC50 value (ng/ml) of chimericllama-human CD70 mAbs and reference mAbs tested in Raji-based co-cultureassay mAb IC50 (ng/ml)  9D1 29  5F4 32  9B2 213  4D2 54  9E1 517  5B2 19SGN70 55 MDX69A7 143 27B3 5 19G10 16  9G2 3 57B6 5 59D10 10

Example 7: Antibody Dependent Cellular Cytoxicity Activity

Antibody Dependent Cellular Cytoxicity (ADCC) was measured using thestandard Cr⁵¹-release assay, which was described by McEarchern et al.(Blood (2007) 109: 1185-1192). Human peripheral blood mononuclear cells(PBMC) were purified from heparinized whole blood by standard ficollseparation and were used as source of NK cells (i.e. effector cells).Blood from several independent donors was used. The cells were suspendedat 2×10⁶/ml in media containing 200 U/ml of human IL-2 (for stimulationof NK cells) and incubated overnight at 37° C. The following day,adherent and non-adherent cells were collected and washed once inculture media. The target cells were 786-O cells (RCC) and were labeledwith Cr⁵¹. Target to effector cell ratio was 1:20 and the incubation wasperformed for 2 hours with the antibody present at differentconcentrations in the culture medium. Chimeric llama-human mAbs as wellas the benchmark antibodies were tested for ADCC activity on 786-O cellsat different concentrations.

The percent lysis was determined by the equation:% Lysis=(sample CPM−spontaneous release CPM)/(maximum releaseCPM−spontaneous release CPM)×100.The results demonstrate that all of the chimeric llama-human mAbs inducelysis of target cells via ADCC activity (FIG. 7). SIMPLE mAb 27B3 has apotency of around 1 μg/ml and therefore is 10 fold more potent than theaffinity variant 9D1 that has a comparable IC50 as benchmark SGN70,which again is 2.5 fold more potent in ADCC than benchmark MDX69A7.SIMPLE antibody 27B3 combines best in class receptor-ligand blockingpotency with superior ADCC potency and therefore has a favourabletherapeutic profile.

Example 8: Complement Dependent Cytoxicity Potency of CD70 Specific mAbs

Complement Dependent Cytotoxicity (CDC) properties of the antibodieswere determined in the standard assay described by McEarchern et al.(Blood (2007) 109: 1185-1192). In a first control experiment, U266 (MM)and MHH-PREB1 (NHL) cells were tested for the presence of CD70 antigenby FACS analysis. In a next experiment, a chimeric llama-human CD70 mAbwas tested for CDC activity on both cell lines at 3 differentconcentrations and in the presence of 3, 6 or 9% human serum (as asource of human complement). It was concluded that the U266 cells givethe best signal/noise ratio in the presence of 9% serum.

In a next experiment, U266 cells were mixed with chimeric llama-humanmAbs (FIG. 8, upper panel) or the reference CD70 mAbs (FIG. 8, lowerpanel) at 0.001-20 μg/ml in the absence or presence of 9% human serumand incubated for 2 h at 37° C. Cells were spun down and PropidiumIodide was added to determine the number of dead cells. Cell lysis wasmeasured in FACS. CDC activity was demonstrated for all chimericllama-human mAbs on U266 cells in the presence of 9% human serum. SIMPLEantibody 27B3 has a similar IC50 as benchmark SGN70, while again MDX69A7is 10 fold less potent.

Example 9: Antibody Dependent Cellular Phagocytosis Potency

To analyze Antibody Dependent Cellular Phagocytosis (ADCP) properties ofthe SIMPLE mAbs the assay described by McEarchern et al. (Blood (2007)109: 1185-1192) was implemented. In this assay human peripheral bloodmononuclear cells (PBMC) were purified from heparinized whole blood bystandard ficoll separation. Blood from different donors were used. Cellswere incubated in RPMI containing 10% FCS in tissue culture flaskovernight at 37° C. Non-adherent cells were removed and adherent cellswere cultured for 15 days in 75 ml X-VIVO 15 medium containing 500 U/mlrhGM-CSF (7.5 μg/75 ml). After 15 days, adherent cells were harvested(monocyte derived macrophages (MDM).

8.0×10⁴ target cells (786-O cells) loaded with the red dye PKH26 wereincubated with mAb for 30 minutes on ice in a V-bottom 96 well plate.Next, 2.0×10⁴ MDM cells were added (target to effector (786-0 to MDM)ratio of 4:1) and after 1 hour of incubation at 37° C., the cells werecentrifuged, resuspended in 100 μl PBS 1% BSA and transferred to aV-bottom 96 well plate. Anti-CD11b monoclonal antibody (FITC conjugated)for staining of macrophages was added for 30 min while keeping theplates on ice, cells were then washed two times with PBS and fixed with1% paraformaldehyde (in PBS). Samples were analysed by FACS, wherephagocytosis was scored on double stained cells. The results are shownin FIG. 9 and these demonstrate that all chimeric llama-human mAbstested are potent in inducing ADCP. SIMPLE antibody 27B3 inducesphagocytosis of up to 50% of the labelled tumour cells by macrophages atconcentrations of 1 μg/ml with an IC50 of around 100 ng/ml (0.4 nM).

Example 10: Internalization of Anti-CD70 mAbs on Tumor Cell Line 786-O

It has been demonstrated that binding of CD70 antibodies to the renalcarcinoma derived cell line 786-O results in the rapid (within 1 hour)internalization of the antibody-receptor complex (Adam et al., Br JCancer (2006) 95:298-306). In order to test for internalization ofSIMPLE antibodies and benchmark mAbs directed against CD70, 786-O cellswere cultured in a 96-well microtiter plate and incubated overnight at37° C. 2.5 μg/ml mAb was added and incubated with the cells for 0-24hours at 37° C. Plates were washed 3 times 5′ with stripping buffer (150mM NaCl, 100 mM Glycine, pH=2.5; coded “IN” representing the amount ofmAb internalized via CD70) or PBS (coded “OUT” and representing theamount of mAb bound to the receptor at the outside of the cell).Subsequently, cells were fixed with 4% paraformaldehyde for 30′ at RT,washed with PBS, and incubated 5′ with 0.2% Triton-X-100 (“IN”) or PBS(“OUT”). Next, cells were washed twice and incubated at RT for 10′ with100 mM glycine followed by a 30′ incubation with PBS+1% BSA. Finallycells were stained with goat anti-human Fc (Jackson immunoresearch109-005-098) and anti-goat IRDYE800 (Li-cor 926-32214) before analysison the Li-Cor Odyssey infrared scanner. The antibody remaining at theoutside of the cell, i.e. non-internalized, is scored as mean MFI OUT inFIG. 10. The results demonstrate that several mAbs remain longer at thesurface of the cells than the other mAbs (higher signal). However, noneof the mAbs internalizes completely: initially (between 0 and 2 hours),mAb internalization goes very fast, but then it seems that a steadystate is reached where 30-40% of the mAb remains at the outside of thecell, even after 24 hours of incubation at 37° C. SIMPLE antibody 27B3internalizes as rapidly as benchmark antibodies SGN70 and MDX69A7. It isinteresting to observe that SIMPLE antibodies 9E1 and 19G10, but alsoother, remain at high levels at the outside of the cells, even after 24hours of incubation (lower panel of FIG. 10). In order to effectivelyinduce ADCC, CDC and ADCP effects it is important that high levels ofCD70 specific antibodies remain on the outside of the targeted cancercells in order to recruit the effector immune cells that are responsiblefor cell killing. On the other hand rapidly internalizing antibodieshave potential as Antibody Drug Conjugates (ADC).

Example 11: In Vivo Efficacy of Chimeric Llama-Human mAbs in a TumourXenograft Model

To establish disseminated disease, 10⁶ Raji cells in 0.2 mL PBS wereinjected into the lateral tail vein of C.B.-17 severe combinedimmunodeficient (SCID) mice (Harlan, Indianapolis, Ind.). Afterinjection, all of the mice were pooled and then placed randomly into thevarious treatment groups, with 9 mice per group. Antibodies wereadministered in 0.5 ml at day 1, 4, 8, 11, 15 after the cellimplantation by intraperitoneal injection. Mice were monitored at leasttwice per week and were sacrificed when they exhibited signs of disease,including weight loss of 15% to 20%, hunched posture, lethargy, cranialswelling, or dehydration. Mice received treatment with 41D12 mAb in adose response. Additionally, a Fc-dead version of 41D12 was tested at 10mg/kg. This Fc-dead version has been described before in the literatureand has no ADCC and CDC activity and much lower ADCP activity(McEarchern et al., Clin Cancer Res (2008) 14(23):7763-72). Results areshown as survival curves in FIG. 11. These data show that the mediansurvival time for the control mice is 27 days. Median survival time wasprolonged to at least 67 days when doses equal to or higher than 0.1mg/kg of 41D12 were administered. The dose of 0.01 mg/kg is stillefficacious. The Fc-dead version of 41D12 has no potency in vivo,illustrating the important of ADCC, CDC and ADCP in this model.

Example 12: Expression of Non-Fucosylated mAbs

Antibodies with reduced amounts of fucosyl residues have beendemonstrated to increase ADCC by 10-1000 fold (Lida et al., Clin CancerRes. (2006) 12(9): 2879-2887). The CHO cell line Ms704-PF and MS705,which lack the fucosyltransferase gene (FUT8, BioWa, Inc.) waselectroporated with a eukaryotic expression vector encoding the heavyand light chain of the chimeric llama-human CD70 mAbs. Drug resistantclones were selected by growth in Excell 325-PF CHO medium. Clones werescreened for IgG production by a standard CD70 binding ELISA.

TABLE 6 sequence of CD70 specific llama-derived VH SEQ SEQ Fab ID IDFramework 1 CDR1 1C2 ELQVVESGGGLVQPGGSLRLSCAASGFTLS 1 NYWMH 10 9D1EVQLVESGGGLVQPGGSLRLSCAASGFTFS 2 VYYMN 11 8B12EVQLVESGGGLVQPGGSLRLSCAASGFTFS 2 VYYMN 11 8C12EVQLVESGGGLVQPGGSLRLSCAASGFTFS 2 VYYMN 11 9E1EVQLQESGGGVVQPGGSLRLSCAASGFTFD 3 DYAMS 12 5F4EVQVQESGGGLVHPGGSLKLSCAASGFTFD 4 TYAMS 13 5B2QVQLVESGGDLVQPGGSLRLSCAASGFTVS 5 NPAMS 14 6D5EVQLVQPGAELRKPGASVKVSCKASGYTFT 6 SYYID 15 4D2QVQLQESGPGLVKPSQTLSLACTVSGGSIT 7 TSYYYWS 16 9A1EVQLVESGGGLVQPGGSLRLSCAASGFTFS 2 SYAMS 17 9G2QVQLVESGGGLMQPGGSLRLSCAASGFTFS 8 SSAMS 18 9B2QLQVVESGGGLMQPGGSLRLSCAASGFTFS 9 GSAMS 19 27B3EVQLVESGGGLVQPGGSLRLSCAASGFTFS 2 VYYMN 11 24E3EVQLVESGGGLVQPGGSLRLSCAASGFTFS 2 VYYMN 11 33D8EVQLVESGGGLVQPGGSLRLSCAASGFTFS 2 VYYMN 11 24F2EVQLVESGGGLVQPGGSLRLSCAASGFTFS 2 VYYMN 11 24B6EVQLQESGGGVVQPGGSLRLSCAASGFTFD 3 DYAMS 12 19G10EVQLQESGGGVVQPGGSLRLSCAASGFTFD 3 DYAMS 12 45B12EVQLVESGGGLVQPGGSLRLSCAASGFTFS 2 AYYMN 20 45D9EVQLVESGGGLVQPGGSLRLSCAASGFTFS 2 AYYMN 20 45F8EVQLVESGGGLVQPGGSLRLSCAASGFTFS 2 AYYMN 20 45Al2EVQLVESGGGLVQPGGSLRLSCAASGFTFS 2 AYYMN 20 45B6EVQLVESGGGLVQPGGSLRLSCAASGFTFS 2 AYYMN 20 57B6QLQLVESGGGLVQPGGSLRLSCAASGFSFS 254 HYAMS 256 59D10EVQLVESGGGLVQPGGSLRLSCAASELSFS 255 ISEMT 257 Framework 2 CDR2 1C2WVRQAPRKGLEWVS 21 TISTDNSRTYYADSVKG 26 9D1 WVRQAPGKGLEWVS 22DINNEGGTTYYADSVKG 27 8B12 WVRQAPGKGLEWVS 22 DINNEGDTTYYADSVKG 28 8C12WVRQAPGKGLEWVS 22 DINNEGDTTYYADSVKG 28 9E1 WVRQAPGKGLEWVS 22SIYMYDSSTYYADSVKG 29 5F4 WVRQAPGKGLEWVS 22 AISWSGGETFYAESMKG 30 5B2WVRQAPGKGLEWVS 22 EITNYGYNRYYADSVKG 31 6D5 WVRQAPGQGLEWMG 23RIDPEDGGTKYAQKFQG 32 4D2 WIRQPPGKGLEWMG 24 AIGSRGSTYYSPSLKT 33 9A1WVRQAPGKGLEWVS 22 DINSGGGSTKYNDSVKG 34 9G2 WVRQAPGKGLEWVS 22SIYSDSSYTYYADSVKS 35 9B2 WVRQAPGKGLEWVS 22 SIYSHSMYTYYADSVKS 36 27B3WVRQAPGKGLEWVS 22 DINNEGGTTYYADSVKG 27 24E3 WVRQAPGKGLEWVS 22DINNEGGTTYYADSVKG 27 33D8 WVRQAPGKGLEWVS 22 DINNEGGTTYYADSVKG 27 24F2WVRQAPGKGLEWVS 22 DINNEGGTTYYADSVKG 27 24B6 WVRQAPGKGLEWVS 22SIYMYDSSTYYADSVKG 29 19G10 WVRQAPGKGLEWVS 22 SIYMYDSSTYYADSVKG 29 45B12WVRQAPGKGLEWIS 25 DINNEGYETYYADSVKG 37 45D9 WVRQAPGKGLEWIS 25DINNEGYETYYADSVKG 37 45F8 WVRQAPGKGLEWIS 25 DINNEGYETYYADSVKG 37 45Al2WVRQAPGKGLEWIS 25 DINNEGYETYYADSVKG 37 45B6 WVRQAPGKGLEWIS 25DINNEGYETYYADSVKG 37 57B6 WVRQAPGKGLEWVS 22 GDNTYDGGTRYQDSVKG 258 59D10WVRQAPGKGLEWVS 22 GISGVTGGSSTSYADSVKG 259 Framework 3 CDR3 1C2RFTISRDHAKNTLILQMNSLKSEDTAVYYCIR 38 GSDYEH 49 9D1RFTISRDNAKNTLTLQMNSLKPEDTALYYCVR 39 DAGYSNHVPIFDS 50 8B12RFTISRDNAKNTLTLQMDSLKPEDTALYYCVR 40 DAGYSNHVPIFDS 50 8C12RFTISRDNAKNTLTLQMDSLKPEDTALYYCVR 40 DAGYSNHVPIFDS 50 9E1RFTISTDNAKNTVYLQMNSLKSEDTAVYYCAK 41 DINRSYGSSWSHFGPIFSS 51 5F4RFTISRNNAKNTLYLQMNSLKSEDTAVYYCAR 42 GMGLAEGLTD 52 5B2RFTISTDNAKNTLYLQMNSLRSEDSAVYYCTA 43 SLGLEYGYGD 53 6D5RVTFTADASTSTAYVELSSLRSEDTAVYYCAS 44 RRRDFDY 54 4D2RTSISRDTSKNQFTLQLSSVTPEDTAVYYCAR 45 VTGEITYNSGSYYYTLNLFDY 55 9A1RFAISRDNAKNTLYLQMNSLKPEDTAVYYCAK 46 EGGSGRYWTNEYDY 56 9G2RFTISTDNAKNTLYLQMNSLKPDDTAVYYCAG 47 SSDYEGSFAS 57 9B2RFTISTDNAKNTLYLQMNSLKPDDTAVYYCAA 342 SSDYEGLFVS 58 27B3RFTISRDNAKNTLTLQMNSLKPEDTALYYCVR 39 DAGYSNHVPIFDS 50 24E3RFTISRDNAKNTLTLQMNSLKPEDTALYYCVR 39 DAGYSNHVPIFDS 50 33D8RFTISRDNAKNTLTLQMNSLKPEDTALYYCVR 39 DAGYSNHVPIFDS 50 24F2RFTISRDNAKNTLTLQMNSLKPEDTALYYCVR 39 DAGYSNHVPIFDS 50 24B6RFTISTDNAKNTVYLQMNSLKSEDTAVYYCAK 41 DINRSYGSSWSHFGPIFSS 51 19G10RFTISTDNAKNTVYLQMNSLKSEDTAVYYCAK 41 DINRSYGSSWSHFGPIFSS 51 45B12RFTISRDNAKNTLTLQMDSLKPEDTARYYCVR 48 DAGYSNHVPIFDS 59 45D9RFTISRDNAKNTLTLQMDSLKPEDTARYYCVR 48 DAGYSNHVQIFDS 59 45F8RFTISRDNAKNTLTLQMDSLKPEDTARYYCVR 48 DAGYSNHVQIFDS 59 45Al2RFTISRDNAKNTLTLQMDSLKPEDTARYYCVR 48 DAGYSNHVQIFDS 59 45B6RFTISRDNAKNTLTLQMDSLKPEDTARYYCVR 48 DAGYSNHVQIFDS 59 57B6RFTISRDNGKNTLYLQMNSLKPEDTAVYYCAK 260 DTGRGIMGEYGMDY 262 59D10RFTISRDNDKNTLYLQMNSLIPEDTAVYYCAT 261 TSGTYYFIPEYEY 263 FRAMEWORK 4 1C2WGQGTQVTVSS 60 9D1 WGQGTQVIVAS 61 8B12 WGQGTQVIVAS 61 8C12 WGQGTQVIVAS61 9E1 WGQGTQVTVSS 60 5F4 WGQGTQVTVSS 60 5B2 WGQGTQVTVSS 60 6D5WGQGTQVTVSS 60 4D2 WGQGTQVTVSS 60 9A1 WGQGTQVTVSS 60 9G2 WGQGTQVTVSS 609B2 WGQGTQVTVSS 60 27B3 WGQGTQVIVAS 61 24E3 WGQGTQVIVAS 61 33D8WGQGTQVIVAS 61 24F2 WGQGTQVIVAS 61 24B6 WGQGTQVTVSS 60 19G10 WGQGTQVTVSS60 45B12 WGQGTQVIVAS 61 45D9 WGQGTQVIVAS 61 45F8 WGQGTQVIVAS 61 45Al2WGQGTQVIVAS 61 45B6 WGQGTQVIVAS 61 57B6 WGKGTLVTVSS 264 59D10WGQGTQVTVSS 60

TABLE 7sequence of CD70 specific llama-derived VL (0 in bold encoded by amberSTOP codon) SEQ SEQ Fab ID ID Framework 1 CDR1 1C2QTVVTQEPSLSVSPGGTVTLTC 62 GLSSGSVTTTNYPG 77 9D1 QAVVTQEPSLSVSPGGTVTLTC63 GLSSGSVTSSHYPG 78 8B12 QTVVTQEPSLSVSPGGTVTLTC 62 GLSSGSVTSSNYPG 798C12 QAVVTQEPSLSVSPGGTVTLTC 63 GLTSGSVTSSNYPD 80 9E1QAVVTQEPSLSVSPGGTVTLTC 63 GLTSGSVTSSNYPD 80 5F4 QAVVTHPPSLSASPGSSVRLTC64 TLISGDNIGGYDIS 81 5B2 QSALTQPPSVSGTLGKTLTISC 65 AGTSSDVGYGNYVS 82 6D5QSALTQPSAVSVSLGQTARITC 66 QGGNARFSSFA 83 4D2 QSVLTQPPSLSASPGSSVRLTC 67TLSSGNSVGNYDIS 84 9A1 QSALTQPSALSVTLGQTAKITC 68 QGGRLGSSYAH 85 9G2QSVVTQPPSLSASPGSSVRLTC 69 TLSSGNSVGNYDIS 84 9B2 QAVLTQPPSLSASPGSSVRLTC70 TLNSANSVGSYDIS 86 27B3 QAVVTQEPSLTVSPGGTVTLTC 71 GLKSGSVTSTNFPT 8724E3 QAVVTQEPSLSVSPGGTVTLTC 63 GLTSGSVTSDNFPV 88 33D8QSALTQPSTVSVSLGQTARITC 72 RGDSLERYGTN 89 24F2 QSALTQPSAVSVSLGQTARITC 66RGDTLRNYHAN 90 24B6 QPVLTQPSAVSVSLGQTARITC 73 QGGYYTH 91 19G10NFMLTQPSAVSVSLGQTARITC 74 QGGYYTH 91 45B12 QAVLTQPSSVSVSLGQTAKITC 75QGGNLGLYGAN 92 45D9 QAVLTQPSSVSVSLGQTAKITC 75 QGGNLWLYGAN 93 45F8QAVLTQPSSVSVSLGQTANITC 76 QGGNLGLYGAN 92 45Al2 QAVVTQEPSLSVSPGGTVTLTC 63GLSSGSATSGNYPE 94 45B6 QAVVTQEPSLSVSPGGTVTLTC 63 GLSSGSVTSSNYPD 95 57B6QTVVTQEPSLSVSPGGTVTLTC 265 GLKSGSVTSSNYPA 267 59D10QSVLTQPPSVSGSPGKTVTISC 266 AGTSSDVGYGYYVS 268 Framework 2 CDR2 1C2WFQQTPGQAPRTLIY 96 STSSRHS 109 9D1 WYQQTPGQAPRLLIF 97 NTNSRHS 110 8B12WYQQTPGQAPRVLIY 98 NTNNRHS 111 8C12 WYQQTPGQAPRLLIY 99 NTNSRHS 110 9E1WYQQTPGQAPRLLIY 99 NTNSRHS 110 5F4 WYQQKTGSPPRYLLY 100 YYSDSYKHQSS 1125B2 WYQQLPGTAPKLLIY 101 RVSTRAS 113 6D5 WYQQKPGQAPVQVIY 102 YNTNRPS 1144D2 WYQQKAGSPPRYLLY 103 YYSDSYKNQGS 115 9A1 WYQQKPGQAPVLVIY 104 GNNYRPS116 9G2 WYQQKAGSPPRYLLY 103 YYSDSVKHQGS 117 9B2 WYQQKAGSPPRYLLY 103YYSDSLSHQGS 118 27B3 WYQQTPGQAPRLLIY 99 NTNTRHS 119 24E3 WYQQTPGQAPRLLIY99 TINSRHS 120 33D8 WYQQKPGQAPVLVIY 104 DDDSRPS 121 24F2 WYRQKPGQAPVLVIY105 GDDIRPS 122 24B6 WYQQKPGQAPVLVIY 104 INNNRPS 123 19G10WYQQKPGQAPVLVIY 104 VNNNRPS 124 45B12 WYQQNPGRAPILLIY 106 GDNYRPL 12545D9 WYQQNPGRAPILLIY 106 GDNQRPL 126 45F8 WYQQNPGRAPILLFY 107 GDNYMPL127 45Al2 WYQQTPGQAPRLIIY 108 NTASRHS 128 45B6 WYQQTPGQAPRLLIY 99NTNSRHS 110 57B6 WYQQTPGQAPRLLIY 99 NTNSRHS 110 59D10 WYQQFPGMAPKLLIY269 DVNKRAS 270 Framework 3 CDR3 1C2 GVPSRFSGSISGNKAALTITGAQPEDEADYYC129 ALEEIGSYTYM 148 9D1 GVPSRFSGSISGNKAALTITGAQPEDEADYYC 129 ALLNIDDGSTM149 8B12 GVPSRYSGSISGNKAALTITGAEPEDEADYYC 130 NLHLGSYTPM 150 8C12GVPSRFSGSISGNKAALTITGAQPEDEADYYC 131 ALYWGYGTNVDV 151 9E1GVPSRFSGSISGNKAALTITGAQPEDEADYYC 129 NLYMGSGGSKV 152 5F4GVPSRFSGSKDASANAGLLLISGLQSEDEADYYC 132 SAYKSGSYKAPV 153 5B2GMPDRFSGSKSGNTASLTISGLQSEDEADYYC 133 ASYTTNNKPV 154 6D5GIPARFSGSSSGGAATLTISGAQAEDEADYYC 134 QSYESGNYV 155 4D2GVPSRFSGSKDPSANAGLLLISGLQAEDEADYYC 135 SVSNSGTYKPV 156 9A1GIPERFSGSSSGDTATLTISGAQAEDEAVYYC 136 QSGSSNTNVM 157 9G2GVPSRFSGSTDASANAGLLLISGLQPEDEADYYC 137 SAYKSGSHV 158 9B2GVPSRFSGSTDASANAGLLLISGLQPEDEADYYC 138 SAYNRGSHV 159 27B3GVPSRFSGSISENKAALTITGAQPEDEAEYFC 139 ALFISNPSVE 160 24E3GVPSRFSGSITGNKAILTITGAQPEDEADYYC 140 ALYLENFANE 161 33D8GIPERFSGSSSGATAALTISGAQAEDEGDYYC 141 QSADSSGNAV 162 24F2GIPERFSGSRLGGTATLTVSGAQAEDEADYYC 142 QSSDSSGYRVV 163 24B6GIPERFSGSISGNTATLTISGAQVEDEADYYC 143 QSGSSSTIPV 164 19G10GIPERFSGSSSGNTATLTISGAQAEDEAAYYC 144 QSGSSSTIPV 164 45B12GIPERFTISKSGGTATLTIDGAQAEDESDYYC 145 QSADYSGNSV 165 45D9GIPERFTISKSGGTATLTIDGAQAEDESDYYC 145 QSADYSGNSV 165 45F8GIPERFTISKSGGTATLTIDGAQAENESDYYC 146 QSSDYPGNSV 166 45Al2GVPGRFSGSISGNKAALTITGAQPEDEADYYC 147 LLYMGGSDFNFV 167 45B6GVPSRFSGSISGNKAALTITGAQPEDEADYYC 129 ALYMGSGSNNVV 168 57B6GVPSRFSGSISGNKAALTITGAQPEDEADYYC 129 ALYMGSGSANAM 271 59D10GIADRFSGSKAGNTASLTISGLQSEDEADYYC 272 ASYRSSANAV 273 Framework 4 1C2FGGGTHLTVLG 169 9D1 FGGGTHLTVLG 169 8B12 FGGGTKLTVLG 170 8C12FGGGTKLTVLG 170 9E1 FGGGTKLTVLG 170 5F4 FGGGTHLTVLG 169 5B2 FGGGTHLTVLG169 6D5 FGGGTTLTVLG 171 4D2 FGGGSKLTVLG 172 9A1 FGGGTHLTVLS 173 9G2FGGGTKLTVLG 170 9B2 FGGGTKLTVLG 170 27B3 FGGGTQLTVLS 174 24E3FGGGTRLTVLG 175 33D8 FGGGTHLTVLG 169 24F2 FGGGTKLTVLG 170 24B6FGGGTKLTVLG 170 19G10 FGGGTKLTVLG 170 45B12 FGGGTKLTVLG 170 45D9FGGGTKLTVLG 170 45F8 FGGGTKLTVLG 170 45Al2 FGGGTKLTVLG 170 45B6FGGGTELTVLG 176 57B6 FGGGTHLTVLG 169 59D10 FGGGTHLTVLG 169

TABLE 8 Full Length llama-derived VH FabFull Length Sequence of VH domain SEQ ID 1C2ELQVVESGGGLVQPGGSLRLSCAASGFTLSNYVVMHWVRQAPRKG 177LEWVSTISTDNSRTYYADSVKGRFTISRDHAKNTLILQMNSLKSEDT AVYYCIRGSDYEHWGQGTQVTVSS9D1 EVQLVESGGGLVQPGGSLRLSCAASGFTFSVYYMNWVRQAPGKG 178LEWVSDINNEGGTTYYADSVKGRFTISRDNAKNTLTLQMNSLKPEDTALYYCVRDAGYSNHVPIFDSWGQGTQVIVAS 8B12EVQLVESGGGLVQPGGSLRLSCAASGFTFSVYYMNWVRQAPGKG 179LEWVSDINNEGDTTYYADSVKGRFTISRDNAKNTLTLQMDSLKPEDTALYYCVRDAGYSNHVPIFDSWGQGTQVIVAS 8C12EVQLVESGGGLVQPGGSLRLSCAASGFTFSVYYMNWVRQAPGKG 179LEWVSDINNEGDTTYYADSVKGRFTISRDNAKNTLTLQMDSLKPEDTALYYCVRDAGYSNHVPIFDSWGQGTQVIVAS 9E1EVQLQESGGGVVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKG 180LEWVSSIYMYDSSTYYADSVKGRFTISTDNAKNTVYLQMNSLKSEDTAVYYCAKDINRSYGSSWSHFGPIFSSWGQGTQVTVSS 5F4EVQVQESGGGLVHPGGSLKLSCAASGFTFDTYAMSWVRQAPGKG 181LEWVSAISWSGGETFYAESMKGRFTISRNNAKNTLYLQMNSLKSEDTAVYYCARGMGLAEGLTDWGQGTQVTVSS 5B2QVQLVESGGDLVQPGGSLRLSCAASGFTVSNPAMSWVRQAPGKG 182LEWVSEITNYGYNRYYADSVKGRFTISTDNAKNTLYLQMNSLRSEDSAVYYCTASLGLEYGYGDWGQGTQVTVSS 6D5EVQLVQPGAELRKPGASVKVSCKASGYTFTSYYIDWVRQAPGQGL 183EWMGRIDPEDGGTKYAQKFQGRVTFTADASTSTAYVELSSLRSED TAVYYCASRRRDFDYWGQGTQVTVSS4D2 QVQLQESGPGLVKPSQTLSLACTVSGGSITTSYYYWSWIRQPPGK 184GLEWMGAIGSRGSTYYSPSLKTRTSISRDTSKNQFTLQLSSVTPEDTAVYYCARVTGEITYNSGSYYYTLNLFDYWGQGTQVTVSS 9A1EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKG 185LEWVSDINSGGGSTKYNDSVKGRFAISRDNAKNTLYLQMNSLKPEDTAVYYCAKEGGSGRYWTNEYDYWGQGTQVTVSS 9G2QVQLVESGGGLMQPGGSLRLSCAASGFTFSSSAMSWVRQAPGKG 186LEWVSSIYSDSSYTYYADSVKSRFTISTDNAKNTLYLQMNSLKPDDTAVYYCAGSSDYEGSFASWGQGTQVTVSS 9B2QLQVVESGGGLMQPGGSLRLSCAASGFTFSGSAMSWVRQAPGK 187GLEWVSSIYSHSMYTYYADSVKSRFTISTDNAKNTLYLQMNSLKPDDTAVYYCAASSDYEGLFVSWGQGTQVTVSS 27B3EVQLVESGGGLVQPGGSLRLSCAASGFTFSVYYMNWVRQAPGKG 178LEWVSDINNEGGTTYYADSVKGRFTISRDNAKNTLTLQMNSLKPEDTALYYCVRDAGYSNHVPIFDSWGQGTQVIVAS 24E3EVQLVESGGGLVQPGGSLRLSCAASGFTFSVYYMNWVRQAPGKG 178LEWVSDINNEGGTTYYADSVKGRFTISRDNAKNTLTLQMNSLKPEDTALYYCVRDAGYSNHVPIFDSWGQGTQVIVAS 33D8EVQLVESGGGLVQPGGSLRLSCAASGFTFSVYYMNWVRQAPGKG 178LEWVSDINNEGGTTYYADSVKGRFTISRDNAKNTLTLQMNSLKPEDTALYYCVRDAGYSNHVPIFDSWGQGTQVIVAS 24F2EVQLVESGGGLVQPGGSLRLSCAASGFTFSVYYMNWVRQAPGKG 178LEWVSDINNEGGTTYYADSVKGRFTISRDNAKNTLTLQMNSLKPEDTALYYCVRDAGYSNHVPIFDSWGQGTQVIVAS 24B6EVQLQESGGGVVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKG 180LEWVSSIYMYDSSTYYADSVKGRFTISTDNAKNTVYLQMNSLKSEDTAVYYCAKDINRSYGSSWSHFGPIFSSWGQGTQVTVSS 19G10EVQLQESGGGVVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKG 180LEWVSSIYMYDSSTYYADSVKGRFTISTDNAKNTVYLQMNSLKSEDTAVYYCAKDINRSYGSSWSHFGPIFSSWGQGTQVTVSS 45B12EVQLVESGGGLVQPGGSLRLSCAASGFTFSAYYMNWVRQAPGKG 188LEWISDINNEGYETYYADSVKGRFTISRDNAKNTLTLQMDSLKPEDTARYYCVRDAGYSNHVQIFDSWGQGTQVIVAS 45D9EVQLVESGGGLVQPGGSLRLSCAASGFTFSAYYMNWVRQAPGKG 188LEWISDINNEGYETYYADSVKGRFTISRDNAKNTLTLQMDSLKPEDTARYYCVRDAGYSNHVQIFDSWGQGTQVIVAS 45F8EVQLVESGGGLVQPGGSLRLSCAASGFTFSAYYMNWVRQAPGKG 188LEWISDINNEGYETYYADSVKGRFTISRDNAKNTLTLQMDSLKPEDTARYYCVRDAGYSNHVQIFDSWGQGTQVIVAS 45A12EVQLVESGGGLVQPGGSLRLSCAASGFTFSAYYMNWVRQAPGKG 188LEWISDINNEGYETYYADSVKGRFTISRDNAKNTLTLQMDSLKPEDTARYYCVRDAGYSNHVQIFDSWGQGTQVIVAS 45B6EVQLVESGGGLVQPGGSLRLSCAASGFTFSAYYMNWVRQAPGKG 188LEWISDINNEGYETYYADSVKGRFTISRDNAKNTLTLQMDSLKPEDTARYYCVRDAGYSNHVQIFDSWGQGTQVIVAS 57B6QLQLVESGGGLVQPGGSLRLSCAASGFSFSHYAMSWVRQAPGKG 274LEWVSGDNTYDGGTRYQDSVKGRFTISRDNGKNTLYLQMNSLKPEDTAVYYCAKDTGRGIMGEYGMDYVVGKGTLVTVSS 59D10EVQLVESGGGLVQPGGSLRLSCAASELSFSISEMTWVRQAPGKGL 275EWVSGISGVTGGSSTSYADSVKGRFTISRDNDKNTLYLQMNSLIPEDTAVYYCATTSGTYYFIPEYEYWGQGTQVTVSS

TABLE 9Full Length llama-derived VL (0 in bold encoded by amber STOP codon) FabFull Length Sequence SEQ ID 1C2QTVVTQEPSLSVSPGGTVTLTCGLSSGSVTTTNYPGWFQQTPGQA 189PRTLIYSTSSRHSGVPSRFSGSISGNKAALTITGAQPEDEADYYCAL EEIGSYTYMFGGGTHLTVLG 9D1QAVVTQEPSLSVSPGGTVTLTCGLSSGSVTSSHYPGWYQQTPGQ 190APRLLIFNTNSRHSGVPSRFSGSISGNKAALTITGAQPEDEADYYCA LLNIDDGSTMFGGGTHLTVLG8B12 QTVVTQEPSLSVSPGGTVTLTCGLSSGSVTSSNYPGWYQQTPGQA 191PRVLIYNTNNRHSGVPSRYSGSISGNKAALTITGAEPEDEADYYCNL HLGSYTPMFGGGTKLTVLG 8C12QAVVTQEPSLSVSPGGTVTLTCGLTSGSVTSSNYPDWYQQTPGQA 192PRLLIYNTNSRHSGVPSRFSGSISGNKAALTITGAQPEDEADYYCAL YWGYGTNVDVFGGGTKLTVLG9E1 QAVVTQEPSLSVSPGGTVTLTCGLTSGSVTSSNYPDWYQQTPGQA 193PRLLIYNTNSRHSGVPSRFSGSISGNKAALTITGAQPEDEADYYCNL YMGSGGSKVFGGGTKLTVLG 5F4QAVVTHPPSLSASPGSSVRLTCTLISGDNIGGYDISWYQQKTGSPP 194RYLLYYYSDSYKHQSSGVPSRFSGSKDASANAGLLLISGLQSEDEADYYCSAYKSGSYKAPVFGGGTHLTVLG 5B2QSALTQPPSVSGTLGKTLTISCAGTSSDVGYGNYVSWYQQLPGTA 195PKLLIYRVSTRASGMPDRFSGSKSGNTASLTISGLQSEDEADYYCA SYTTNNKPVFGGGTHLTVLG 6D5QSALTQPSAVSVSLGQTARITCQGGNARFSSFAWYQQKPGQAPV 196QVIYYNTNRPSGIPARFSGSSSGGAATLTISGAQAEDEADYYCQSY ESGNYVFGGGTTLTVLG 4D2QSVLTQPPSLSASPGSSVRLTCTLSSGNSVGNYDISWYQQKAGSP 197PRYLLYYYSDSYKNQGSGVPSRFSGSKDPSANAGLLLISGLQAEDEADYYCSVSNSGTYKPVFGGGSKLTVLG 9A1QSALTQPSALSVTLGQTAKITCQGGRLGSSYAHWYQQKPGQAPVL 198VIYGNNYRPSGIPERFSGSSSGDTATLTISGAQAEDEAVYYCQSGS SNTNVMFGGGTHLTVLS 9G2QSVVTQPPSLSASPGSSVRLTCTLSSGNSVGNYDISWYQQKAGSP 199PRYLLYYYSDSVKHQGSGVPSRFSGSSDASANAGLLLISGLQPEDE ADYYCSAYKSGSHVFGGGTKLTVLG9B2 QAVLTQPPSLSASPGSSVRLTCTLNSANSVGSYDISWYQQKAGSP 200PRYLLYYYSDSLSHQGSGVPSRFSGSTDASANAGLLLISGLQPEDE ADYYCSAYNRGSHVFGGGTKLTVLG27B3 QAVVTQEPSLTVSPGGTVTLTCGLKSGSVTSTNFPTWYQQTPGQA 201PRLLIYNTNTRHSGVPSRFSGSISENKAALTITGAQPEDEAEYFCAL FISNPSVEFGGGTQLTVLS 24E3QAVVTQEPSLSVSPGGTVTLTCGLTSGSVTSDNFPVWYQQTPGQA 202PRLLIYTINSRHSGVPSRFSGSITGNKAILTITGAQPEDEADYYCALY LENFANEFGGGTRLTVLG 33D8QSALTQPSTVSVSLGQTARITCRGDSLERYGTNWYQQKPGQAPVL 203VIYDDDSRPSGIPERFSGSSSGATAALTISGAQAEDEGDYYCQSAD SSGNAVFGGGTHLTVLG 24F2QSALTQPSAVSVSLGQTARITCRGDTLRNYHANWYRQKPGQAPVL 204VIYGDDIRPSGIPERFSGSRLGGTATLTVSGAQAEDEADYYCQSSD SSGYRVVFGGGTKLTVLG 24B6QPVLTQPSAVSVSLGQTARITCQGGYYTHWYQQKPGQAPVLVIYIN 205NNRPSGIPERFSGSISGNTATLTISGAQVEDEADYYCQSGSSSTIPV FGGGTKLTVLG 19G10NFMLTQPSAVSVSLGQTARITCQGGYYTHWYQQKPGQAPVLVIYV 206NNNRPSGIPERFSGSSSGNTATLTISGAQAEDEAAYYCQSGSSSTI PVFGGGTKLTVLG 45B12QAVLTQPSSVSVSLGQTAKITCQGGNLGLYGANWYQQNPGRAPIL 207LIYGDNYRPLGIPERFTISKSGGTATLTIDGAQAEDESDYYCQSADY SGNSVFGGGTKLTVLG 45D9QAVLTQPSSVSVSLGQTAKITCQGGNLWLYGANWYQQNPGRAPIL 208LIYGDNORPLGIPERFTISKSGGTATLTIDGAQAEDESDYYCQSADY SGNSVFGGGTKLTVLG 45F8QAVLTQPSSVSVSLGQTANITCQGGNLGLYGANWYQQNPGRAPIL 209LFYGDNYMPLGIPERFTISKSGGTATLTIDGAQAENESDYYCQSSDY PGNSVFGGGTKLTVLG 45A12QAVVTQEPSLSVSPGGTVTLTCGLSSGSATSGNYPEWYQQTPGQ 210APRLIIYNTASRHSGVPGRFSGSISGNKAALTITGAQPEDEADYYCL LYMGGSDFNFVFGGGTKLTVLG45B6 QAVVTQEPSLSVSPGGTVTLTCGLSSGSVTSSNYPDWYQQTPGQA 211PRLLIYNTNSRHSGVPSRFSGSISGNKAALTITGAQPEDEADYYCAL YMGSGSNNVVFGGGTELTVLG57B6 QTVVTQEPSLSVSPGGTVTLTCGLKSGSVTSSNYPAWYQQTPGQA 276PRLLIYNTNSRHSGVPSRFSGSISGNKAALTITGAQPEDEADYYCAL YMGSGSANAMFGGGTHLTVLG59D10 QSVLTQPPSVSGSPGKTVTISCAGTSSDVGYGYYVSWYQQFPGMA 277PKLLIYDVNKRASGIADRFSGSKAGNTASLTISGLQSEDEADYYCAS YRSSANAVFGGGTHLTVLG

Example 13: Germlining of Anti-CD70 mAb 27B3

Human germline genes segments with the same canonical fold structure andthe highest amino acid sequence identity to the VH and VL regions of mAb27B3 were identified by comparison with known human germline genesequences. The closest human VH and VL regions to mAb 27B3 was humanVH3-48 (89.7% FR identity) and human VL8-61 (86.1% FR identity),respectively. The closest human germline JH and JL human germline genesegment sequences were JH5 and JL7, respectively. Sequence alignment ofthe VH and VL regions of mAb 27B3 with the closest human germline VH andVL sequences are set forth in FIG. 12. Comparison of the V regions ofSIMPLE antibody 27B3 and human germline sequences identified 12candidate humanizing mutations in the VH region and 13 in the VL region(2 in CDR1 and 1 in CDR2). An overview of the humanizing mutations inthe VH and VL sequences of the 27B3, together with the needed librarysizes to cover the introduced diversity, is set forth in Table 10 and11.

TABLE 10 Targeted mutations in the 27B3 VH amino acid sequence. HumanHuman Human germline Kabat Camelid germline aa germline aa aa Positionaa option 1 option 2 option 3 Probability 46 E V 0.50 74 A S 0.50 77 T S0.50 79 T Y 0.50 83/84 KP KT RA 0.33 89 L V 0.50 93/94 AR AK VR 0.33 108Q L T M 0.25 110 I T 0.50 112 A S 0.50 4608¶ ¶ denotes the library sizeneeded to cover all potential mutants. Mutations included in library

TABLE 11 Targeted mutations in the 27B3 VL amino acid sequence. HumanHuman germline Human germline Kabat Camelid aa germline aa aa Positionaa option 1 option 2 option 3 Probability 2 A T 0.50 11-12 LT FS 0.5026* K T 0.50 30* T D 0.50 46 L T 0.50 53* T S 0.50 60 S D 0.50 61 R C0.50 67/68 SE LG 0.50 80/81/84/85 PEDEAE PEDESD ADDESD ADDEAE 0.25 87 FY 0.50 4096¶ ¶ denotes the library size needed to cover all potentialmutants. *denotes mutations in the CDR1 or CDR2 regions.

Germlined libraries of VH_27B3 and VL_27B3 encompassing all combinationsof humanizing mutations were created by PCR based assembly (see e.g.,Stemmer et al., Gene (1995) 164: 49-53). Overlapping oligonucleotideswith specific mutations on certain positions were assembled by PCR. Thelibrary contained both human and llama amino acids at each mutatedposition to prevent complete loss of binding in the event that the wildtype llama residue was critical for high affinity binding (see e.g.,Baca et al., J. Biol. Chem. (1997) 272: 10678-10684; Tsurushita et al.,J. Immunol. Methods (2004) 295: 9-19). The VH library contained about1×10¹⁰ clones and the VL library about 8×10⁹ clones. The VH and VLlibraries were combined and reformatted into a single Fab library with asize of 1×10¹⁰ clones (91% with full length Fab insert) suitable forphage display screening. To required diversity to cover all possiblemutations as mentioned in tables 10 and 11 was exceeded in the primaryheavy chain and light chain libraries, but also the combined Fab librarywas large enough to cover all possible permutations (1.89×10⁷).

Example 14: Selection of Germlined 27B3 Fabs with High Human FR Identity

Phage display was used to select for germlined 27B3 Fabs with both ahigh affinity for CD70 and high human FR identity. Specifically,Flag-TNC-CD70 was biotinylated and was incubated at variousconcentrations with different amounts of Fab expressing phage. Complexesof phage and Flag-TNC-CD70 were then captured on a streptavidin-coatedmicrotiter plate and washed with non-biotinylated Flag-TNC-CD70 at 37°C. Phage were eluted with Trypsin and used for infection of TG1 cells.Five rounds of selection were performed, with the wash stringencyincreased in each round. Details of the conditions used for each roundof selection are set forth in Table 12.

TABLE 12 Conditions for selection of germlined 27B3 Fabs Phage Washinput Washing temper- Washing Round (μl) Antigen [pM] time ature antigen1 10 24000-2400- 1 hour RT PBS 240 2 1 2400-240-24 ON 37° C. 24 nM CD703 0.1 240-24-2.4 ON 37° C. 2.4 nM CD70  4 0.01 24-2.4-0.24 3 days 37° C.240 pM CD70  5 0.1 2.4-0.24-0.024 6 days 37° C. 24 pM CD70

Phage eluted from rounds 3, 4 and 5 of selection were transfected intoTG1 and plated clonally; individual clones were picked randomly forfurther analysis. Periplasmic fractions containing Fabs were preparedfrom IPTG induced small scale cultures the off rate of each Fab wasdetermined using a CD70 coated CM5 chip in a Biacore binding assay.Clones with Fab having off rates similar to that of 27B3 were sequenced.Off rates for clones with a total human FR sequence identity above 94%are set forth in Table 13, along with the percentage human FR identityof the individual clone (VH, VL and total).

The complete amino acid sequences of the sequenced Fab clones are setforth in tables 14 and 15 and CDR amino acid sequence variants of 27B3are set forth in table 16. Inspection of these sequences reveals thatframework 1 of the light chain often has extra (non human) mutations inthe primer region at amino acid 2 (A instead of T). Variants of clones35G3, 40F1 and 39C3 (clones 53C1, 53B1 and 53E1, respectively) were madethat have the corresponding human amino acid at position 2. Moreover,clone 41D12 was further germlined by combining the heavy chain of 41D12with the light chain of 40F1 (53A2) or with the light chain of 53B1(53H1).

TABLE 13 Off rate of germlined 27B3 Fab clones with more than 94% totalhuman FR identity. % FR % FR Total Fab identity for identity for % FRIsolation Off rate Clone VL VH identity frequency [10⁻⁴ s⁻¹] 27B3 86.089.5 87.9 1.4 36A9 97.5 92.0 94.5 1 1.3 53F1 97.5 92.0 94.5 1 2.3 36D694.9 94.3 94.6 2 1.4 53G1 96.2 94.0 95.0 1 2.0 35G3 94.9 95.4 95.2 1 1.735F6 94.9 95.4 95.2 1 ND 36G2 93.6 96.6 95.2 7 1.1 39D5 93.7 96.6 95.2 21.2 42D12 93.7 96.6 95.2 2 1.6 35G1 97.5 94.3 95.7 1 2.3 41D12 92.4 98.995.9 4 2.3 41H8 94.9 97.0 96.0 1 2.2 35G2 96.2 96.6 96.4 1 1.7 40F1 94.998.0 96.6 1 1.6 39C3 97.5 97.7 97.6 6 2.5

TABLE 14AVH amino acid sequences of germlined 27B3 clones with more than 94% totalhuman identity, with sequence identifiers broken down by FR and CDR.Humanizing mutations are italicized and primer-introduced mutations are bolded.HEAVY CHAIN CLONE FRAMEWORK 1 CDR1 FRAMEWORK 2 27B3EVQLVESGGGLVQPGGSLRLSCAASGFTFS VYYMN WVRQAPGKGLEWVS SEQ ID NO: 2SEQ ID NO: 11 SEQ ID NO: 22 36A9 EVQLVESGGGLVQPGGSLRLSCAASGFTFS VYYMNWVRQAPGKGLEWVS SEQ ID NO: 2 SEQ ID NO: 11 SEQ ID NO: 22 53F1EVQLVESGGGLVQPGGSLRLSCAASGFTFS VYYMN WVRQAPGKGLEWVS SEQ ID NO: 2SEQ ID NO: 11 SEQ ID NO: 22 36D6 EVQLVESGGGLVQPGGSLRLSCAASGFTFS VYYMNWVRQAPGKGLEWVS SEQ ID NO: 2 SEQ ID NO: 11 SEQ ID NO: 22 53G1EVQLVESGGLVQPGGSLRLSCAASGFTFS VYYMN WVRQAPGKGLEWVS SEQ 12 NO: 2SEQ 12 NO: 11 SEQ ID NO: 22 35G3 EVQLVESGGGLVQPGGSLRLSCAASGFTFS VYYMNWVRQAPGKGLEWVS SEQ ID NO: 2 SEQ ID NO: 11 SEQ ID NO: 22 53C1EVQLVESGGGLVQPGGSLRLSCAASGFTFS VYYMN WVRQAPGKGLEWVS SEQ ID NO: 2SEQ ID NO: 11 SEQ ID NO: 22 35F6 EVQLVESGGGLVQPGGSLRLSCASSGFTFS VYYMNWVRQAPGKGLEWVS SEQ ID NO: 274 SEQ ID NO: 11 SEQ ID NO: 22 36G2EVQLVESGGGLVQPGGSLRLSCAASGFTFS VYYMN WVRQAPGKGLEWVS SEQ ID NO: 2SEQ ID NO: 11 SEQ ID NO: 22 39D5 EVQLVESGGGLVQPGGSLRLSCAASGFTFS GYYMNWVRQAPGKGLEWVS SEQ ID NO: 2 SEQ ID NO: 248 SEQ ID NO: 22 42D12EVQLVESGGGLVQPGGSLRLSCAASGFTFS VYYMN WVRQAPGKGLEWVS SEQ ID NO: 2SEQ ID NO: 11 SEQ ID NO: 22 35G1 EVQLVESGGGLVQPGGSVRLSCAASGFTFS VYYMNWVRQAPGKGLEWVS SEQ ID NO: 2 SEQ ID NO: 11 SEQ ID NO: 22 41D12EVQLVESGGGLVQPGGSLRLSCAASGFTFS VYYMN WVRQAPGKGLEWVS SEQ ID NO: 2SEQ ID NO: 11 SEQ ID NO: 22 41H8 EVQLVESGGGLVQPGGSLRLSCAASGFTFS VYYMNWVRQAPGKGLEWVS SEQ ID NO: 2 SEQ 12 NO: 11 SEQ ID NO: 22 35G2EVQLVESGGGLVQPGGSLRLSCAASGFTFS VYYMN WVRQAPGKGLEWVS SEQ ID NO: 2SEQ ID NO: 11 SEQ ID NO: 22 40F1 EVQLVESGGGLVQPGGSLRLSCAASGFTFS VYYMNWVRQAPGKGLEWVS SEQ ID NO: 2 SEQ ID NO: 11 SEQ ID NO: 22 53B1EVQLVESGGGLVQPGGSLRLSCAASGFTFS VYYMN WVRQAPGKGLEWVS SEQ ID NO: 2SEQ ID NO: 11 SEQ ID NO: 22 39C3 EVQLVASGGGLVQPGGSLRLSCAASGFTFS VYYMNWVRQAPGKGLEWVS SEQ ID NO: 275 SEQ ID NO: 11 SEQ ID NO: 22 53E1EVQLVASGGGLVQPGGSLRLSCAASGFTFS VYYMN WVRQAPGKGLEWVS SEQ ID NO: 275SEQ ID NO: 11 SEQ ID NO: 22 53H1 EVQLVESGGGLVQPGGSLRLSCAASGFTFS VYYMNWVRQAPGKGLEWVS SEQ ID NO: 2 SEQ ID NO: 11 SEQ ID NO: 22 53A2EVQLVESGGGLVQPGGSLRLSCAASGFTFS VYYMN WVRQAPGKGLEWVS SEQ ID NO: 2SEQ ID NO: 11 SEQ ID NO: 22 HEAVY CHAIN CLONE CDR2 FRAMEWORK 3 CDR3FRAMEWORK 4 27B3 DINNEGGTTYYADSVKG RFTISRDNAKNTLTIQMNSLKPEDTALYYCVRDAGYSNHVPIFDS WGQGTQVIVAS SEQ ID NO: 27 SEQ ID NO: 39 SEQ ID NO: 50SEQ ID NO: 61 36A9 DINNEGGTTYYADSVKG RFTISRDNSKNTLTLQMNSLKPEDTAVYYCVRDAGYSNHVPIFDS WGQGTQVIVSS SEQ ID NO: 27 SEQ ID NO: 350 SEQ ID NO: 50SEQ ID NO: 287 53F1 DINNEGGTTYYADSVKG RFTISRDNAKNTLTLQMNSLKPEDTAVYYCVRDAGYSNHVPIFDS WGQGTQVTVAS SEQ ID NO: 27 SEQ ID NO: 351 SEQ 12 NO: 50SEQ ID NO: 288 36D6 DINNEGGTTYYADSVKG RFTISRDNAKNTLYLQMNSLKPEDTALYYCVRDAGYSNHVPIFDS WGQGTTVTVSS SEQ ID NO: 27 SEQ ID NO: 278 SEQ ID NO: 50SEQ ID NO: 289 53G1 DINNEGGTTYYADSVKG RFTISRDNAKNTLYLQMNSLKPEDTAVYYCVRDAGYSNHVPIFDS WGQGTTVIVSS SEQ ID NO: 27 SEQ ID NO: 279 SEQ ID NO: 50SEQ ID NO: 290 35G3 DINNEGGTTYYADSVKG RFTISRDNSKNSLTIQMNSLKPEDTAVYYCARDAGYSNHVPIFDS WGQGTLVTVSS SEQ ID NO: 27 SEQ ID NO: 280 SEQ ID NO: 50SEQ ID NO: 293 53C1 DINNEGGTTYYADSVKG RFTISRDNSKNSLTLQMNSLKPEDTAVYYCARDAGYSNHVPIFDS WGQGTLVTVSS SEQ ID NO: 27 SEQ ID NO: 280 SEQ ID NO: 50SEQ ID NO: 293 35F6 DINNEGGTTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCVRDAGYSNHVPIFDS WGQGTTVTVAS SEQ ID NO: 27 SEQ ID NO: 281 SEQ ID NO: 50SEQ ID NO: 291 36G2 DINNEGGTTYYADSVKG RFTISRDNAKNSLTLQMNSLRAEDTAVYYCVRDAGYSNHVPIFDS WGQGTQVTVSS SEQ ID NO: 27 SEQ ID NO: 282 SEQ ID NO: 50SEQ ID NO: 292 39D5 DINNEGGTTYYADSVKG RFTISRDNAKNTLYLQMNSLRAEDTAVYYCVRDAGYSNHVPIFDS WGQGTTVTVAS SEQ ID NO: 27 SEQ ID NO: 283 SEQ ID NO: 50SEQ ID NO: 291 42D12 DINNEGGTTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCVRDAGYSNHVPIFDS WGQGTQVTVSS SEQ ID NO: 27 SEQ ID NO: 281 SEQ ID NO: 50SEQ ID NO: 292 35G1 DINNEGGTTYYADSVKG RFTISRDNAKNTLYLQMNSLKPFDTAVYYCARDAGYSNHVPIFDS WGQGTLVTVAS SEQ ID NO: 27 SEQ ID NO: 284 SEQ ID NO: 50SEQ ID NO: 294 41D12 DINNEGGITYYADSVKG RFTISRDNSKNSLYLQMNSLRAEDTAVYYCARDAGYSNHVPIFDS WGQGTLVTVSS SEQ ID NO: 27 SEQ ID NO: 285 SEQ ID NO: 50SEQ ID NO: 293 41H8 DINNEGGTTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDAGYSNHVPIFDS WGQGTQVTVAS SEQ ID NO: 27 SEQ ID NO: 286 SEQ ID NO: 50SEQ ID NO: 288 35G2 DINNEGGTTYYADSVKG RFTISRDNAKNTLYLQMNSLKPEDTAVYYCARDAGYSNHVPIFDS WGQGTLVTVSS SEQ ID NO: 27 SEQ ID NO: 284 SEQ ID NO: 50SEQ ID NO: 293 40F1 DINNEGGATYYADSVKG RFTISRDNSKNSLYLQMNSLRAEDTAVYYCARDAGYSNHVPIFDS WGQGTQVTVSS SEQ ID NO: 249 SEQ ID NO: 285 SEQ ID NO: 50SEQ ID NO: 292 53B1 DINNEGGATYYADSVKG RFTISRDNSKNSLYLQMNSLRAEDTAVYYCARDAGYSNHVPIFDS WGQGTQVTVSS SEQ ID NO: 249 SEQ ID NO: 285 SEQ ID NO: 50SEQ ID NO: 292 39C3 DINNEGGITYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDAGYSNHVPIFDS WGQGTTVTVSS SEQ ID NO: 27 SEQ ID NO: 286 SEQ ID NO: 50SEQ ID NO: 289 53E1 DINNEGGTTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDAGYSNHVPIFDS WGQGTTVTVSS SEQ ID NO: 27 SEQ ID NO: 286 SEQ ID NO: 50SEQ ID NO: 289 53H1 DINNEGGTTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDAGYSNHVPIFDS WGQGTLVTVSS SEQ ID NO: 27 SEQ ID NO: 285 SEQ ID NO: 50SEQ ID NO: 293 53A2 DINNEGGTTYYADSVKG RFTISRDNSKNSLYLQMNSLRAEDTAVYYCARDAGYSNHVPIFDS WGQGTLVTVSS SEQ ID NO: 27 SEQ ID NO: 285 SEQ ID NO: 50SEQ ID NO: 293

TABLE 14BVH amino acid sequences of germlined 27B3 clones with more than 94% totalhuman identity, with sequence identifiers for the fullVH amino acid sequence. Humanizing mutations are italicized and primer-introduced mutations are bolded. SEQ HEAVY ID CHAIN NO: CLONEFULL VH SEQUENCE 178 27B3EVQLVESGGGLVQPGGSLRLSCAASGETESVYYMNWVRQAPGKGLEWVSDINNEGGTTYYADSVKGRFTISRDNAKNTLTLQMNSLKPEDTALYYCVRDAGYSNHVPIFDSWGQGTQVIVAS 212 36A9EVQLVESGGGLVQPGGSLRLSCAASGFTESVYYMNWVRQAPGKGLEWVSDINNEGGTTYYADSVKGRFTISRDNSKNTLTLQMNSLKPEDTAVYYCVRDAGYSNHVPIFDSWGQGTQVIVSS 213 53F1EVQLVESGGGLVQPGGSLRLSCAASGFIFSVYYMNWVRQAPGKGLEWVSDINNEGGTTYYADSVKGRFTISRDNAKNTLTLQMNSLKPEDTAVYYCVADAGYSNHVPIFDSWGQGTQVTVAS 214 36D6EVQLVESGGGLVQPGGSLRLSCAASGFTFSVYYMNWVRQAPGKGLEWVSDINNEGGTTYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTALYYCVRDAGYSNHVPIFDSWGQGTTVTVSS 215 53G1EVQLVESGGGLVQPGGSLRLSCAASGFTFSVYYMNWVRQAPGKGLEWVSDTNNEGGTTYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCVRDAGYSNHVPIFDSWGQGTTVIVSS 216 35G3EVQLVESGGGLVQPGGSLRLSCAASGFTFSVYYMNWVRQAPGKGLEWVSDINNEGGTTYYADSVKGRFTISRDNSKNSLTLQMNSLKPEDTAVYYCARDAGYSNHVPIFDSWGQGTLVTVSS 217 53C1EVQLVESGGGLVQPGGSLRLSCAASGFTFSVYYMNWRQAPGKGLEWVSDINNEGGTTYYADSVKGRFTISRDNSKNSLTLQMNSLKPEDTAVYYCARDAGYSNHVPIFDSWGQGTLVTVSS 218 35F6EVQLVESGGGLVQPGGSLRLSCASSGFTFSVYYMNWVRQAPGKGLEWVSDINNEGGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVRDAGYSNHVPIFDSWGQGTTVTVAS 219 36G2EVQLVESGGGLVQPGGSLRLSCAASGETESVYYMNWVRQAPGKGLEWVSDINNEGGTTYYADSVKGRFTISRDNSKNSLTLQMNSLRAEDTAVYYCVRDAGYSNHVPIFDSWGQGTQVTVSS 220 39D5EVQLVESGGGLVQPGGSLRLSCAASGFTFSVYYMNWRQAPGKGLEWVSDINNEGGTTYYADSVKGRFTISRDNAKNTLTLYMNSLRAEDTAVYYCVRDAGYSNHVPIFDSWGQGTTVTVAS 221 42D12EVQLVESGGGLVQPGGSLRLSCAASGFTFSVYYMNWRQAPGKGLEWVSDINNEGGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVRDAGYSNHVPIFDSWGQGTQVTVSS 222 35G1EVQLVESGGGLVQPGGSVRLSCAASGFTFSVYYMNWVRQAPGKGLEWVSDINNEGGTTYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCARDAGYSNHVPIFDSWGQGTLVTVAS 223 41D12EVQLVESGGGLVQPGGSLRLSCAASGFTFSVYYMNWVRQAPGKGLEWVSDINNEGGTTYYADSVKGRFTISRDNSKNSLYLQMNSLRAEDTAVYYCARDAGYSNHVPIFDSWGQGTLVTVSS 224 41H8EVQLVESGGGLVQPGGSLRLSCAASGFTFSVYYMNWVRQAPGKGLEWVSDINNEGGTTYYADSVKGRFTTSRDNSKNTLYLQMNSLRAFDTAVYYCARDAGYSNHVPIFDSWGQGTQVTVAS 225 35G2EVQLVESGGGLVQPGGSLRLSCAASGFTFSVYYMNWVRQAPGKGLEWVSDINNEGGTTYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCARDAGYSNHVPIFDSWGQGTLVTVSS 226 40F1EVQLVESGGGLVQPGGSLRLSCAASGFTFSVYYMNWVRQAPGKGLEWVSDINNEGGATYYADSVKGRFTISRDNSKNSLYLQMNSLRAEDTAVYYCARDAGYSNHVPIFDSWGQGTQVTVSS 227 53B1EVQLVESGGGLVQPGGSLRLSCAASGFTFSVYYMNWRQAPGKGLEWVSDINNEGGATYYADSVKGRFTISRDNSKNSLYTLQMNSLRAFDTAVYYCARDAGYSNHVPIFDSWGQGTQVTVSS 228 39C3EVQLVASGGGLVQPGGSLRLSCAASGFTFSVYYMNWRQAPGKGLEWVSDINNEGGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDAGYSNHVPIFDSWGQGTTVTVSS 229 53F1EVQLVASGGGLVQPGGSLRLSCAASGFTFSVYYMNWVRQAPGKGLEWVSDINNEGGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDAGYSNHVPIFDSWGQGTTVTVSS 223 53H1EVQLVESGGGLVQPGGSLRLSCAASGFTFSVYYMNWVRQAPGKGLFWVSDINNEGGTTYYADSVKGRFTISRDNSKNSLYLQMNSLRAEDTAVYYCARDAGYSNHVPIFDSWGQGTLVTVSS 223 53A2EVQLVESGGGLVQPGGSLRLSCAASGFTFSVYYMNWVRQAPGKGLEWVSDINNEGGTTYYADSVKGRFTISRDNSKNSLYLQMNSLRAEDTAVYYCARDAGYSNHVPIFDSWGQGTLVTVSS

TABLE 15AVL amino acid sequences of germline 27B3 clones with more than 94% totalhuman identity, with sequence identifiers brokendown by FR and CDR. Humanizing mutationsare italicized and primer-introduced mutations are bolded. LIGHT CHAINCLONE FRAMEWORK 1 CDR1 FRAMEWORK 2 27B3 QAVVTQEPSLIVSPGGTVTLTCGLKSGSVISTNEPT WYQQTPGQAPRLLIY SEQ ID NO: 71 SEQ ID NO: 87 SEQ ID NO: 9936A9 QTVVTQEPSFSVSPGGTVTLTC GLKSGSVTSDNEPT WYQQTPGQAPRLLIYSEQ ID NO: 294 SEQ ID NO: 250 SEQ ID NO: 99 53F1 QTVVTQEPSFSVSPGGTVTLTCGLKSGSVTSDNEPT WYQQTPGQAPRLLIY SEQ ID NO: 295 SEQ ID NO: 250SEQ ID NO: 99 36D6 QTVVTQEPSLTVSPGGTVTLTC GLKSGSVTSTNFPT WYQQTPGQAPPLLIYSEQ ID NO: 296 SEQ ID NO: 87 SEQ ID NO: 99 53G1 QTVVTQEPSFSVSPGGTVTLTCGLTSGSVTSTNEPT WYQQTPGQAPRLLIY SEQ ID NO: 294 SEQ ID NO: 251SEQ ID NO: 99 35G3 QAVVTQEPSFSVSPGGTVTLTC GLKSGSVTSDNFPT WYQQTPGQAPRLLIYSEQ ID NO: 295 SEQ ID NO: 250 SEQ ID NO: 99 53C1 QTVVTQEPSFSVSPGGTVTLTCGLKSGSVTSDNFPT WYQQTPGQAPRLLIY SEQ ID NO: 294 SEQ ID NO: 250SEQ ID NO: 99 35F6 QTVVTQFPSFSVSPGGTVTLTC GLTSGSVTSDNFPT WYQQTPGQAPPLLIYSEQ ID NO: 294 SEQ ID NO: 252 SEQ ID NO: 99 36G2 QAVVTQEPSLTVSPGGTVTLTCGLKSGSVTSDNFPT WYQQTPGQAPRLLIY SEQ ID NO: 71 SEQ ID NO: 250SEQ ID NO: 99 39D5 QTVVTQEPSLTVSPGGTVTLTC GV TSGSVTSDNFPTWYQQTPGQAPRLLIY SEQ ID NO: 296 SEQ ID NO: 253 SEQ ID NO: 99 42D12QAVVTQEPSFSVSPGGTVTLTC GLTSGSVTSTNFPT WYQQTPGQAPRLLIY SEQ ID NO: 295SEQ ID NO: 251 SEQ ID NO: 99 35G1 QTVVTQEPSFSVSPGGTVTLTC GLKSGSVTSDNFPTWYQQTPGQAPRLLIY SEQ ID NO: 294 SEQ ID NO: 250 SEQ ID NO: 99 41D12QAVVTQEPSLTVSPGGTVTLTC GLKSGSVTSDNFPT WYQQTPGQAPRLLIY SEQ ID NO: 71SEQ ID NO: 250 SEQ ID NO: 99 41H8 QAVVTQEPSFSVSPGGTVTLTC GLTSGSVTSTNFPTWYQQTPGQAPRLLIY SEQ ID NO: 295 SEQ ID NO: 251 SEQ ID NO: 99 35G2QTVVTQFPSFSVSPGGTVTLTC GLKSGSVTSTNFPT WYQQTPGQAPRILIY SEQ ID NO: 294SEQ ID NO: 87 SEQ ID NO: 99 40F1 QAVVTQEPSFSVSPGGTVTLTC GLKSGSVTSDNFPTWYQQTPGQAPRLLIY SEQ ID NO: 295 SEQ ID NO: 250 SEQ ID NO: 99 53B1QTVVTQEPSFSVSPGGTVTLTC GLKSGSVTSDNFPT WYQQTPGQAPRLLIY SEQ ID NO: 294SEQ ID NO: 250 SEQ ID NO: 99 39C3 QAVVTQEPSFSVSPGGTVTLTC GLKSGSVTSDNFPTWYQQTPGQAPRLLIY SEQ ID NO: 295 SEQ ID NO: 250 SEQ ID NOL: 99 53E1QTVVTQEPSFSVSPGGTVTLTC GLKSGSVTSDNFPT WYQQTPGQAPALLIY SEQ ID NO: 294SEQ ID NO: 250 SEQ ID 110: 99 53H1 QTVVTQEPSFSVSPGGIVTLTC GLKSGSVISDNEPTWYQQTPGQAPRLLIY SEQ ID NO: 294 SEQ ID NO: 250 SEQ ID NO: 99 53A2QAVVIQEPSFSVSPGGTVTLTC GLKSGSVTSDNFPT WYQQTPGQAPALLIY SEQ ID NO: 295SEQ ID NO: 250 SEQ ID NO: 99 LIGHT CHAIN CLONE CDR2 FRAMEWORK 3 CDR3FRAMEWORK 4 27B3 NTNTRHS GVPSRFSGSISENKAALTITGAQPEDEAEYFC ALFISNPSVEFGGGTQLTVLS SEQ ID NO: 119 SEQ ID NO: 139 SEQ ID NO: 160 SEQ ID 510: 17436A9 NTNTRHS GVPDRFSGSILGNKAALTITGAQADDESDYFC ALFISNPSVE FGGGTQLTVLGSEQ ID NO: 119 SEQ ID NO: 297 SEQ ID NO: 160 SEQ ID NO: 309 53F1 NTNTRHSGVPDRFSGSILGNKAALTITGAQADDESDYFC ALFISNPSVE FGGGTQLTVLG SEQ ID NO: 119SEQ ID NO: 297 SEQ ID NO: 160 SEQ ID NO: 309 36D6 NTNSRHSGVPDRFSGSILGNKAALTITGAQADDESDYFC ALFISNPSVE FGGGTQLTVLG SEQ ID NO: 110SEQ ID NO: 297 SEQ ID NO: 160 SEQ ID NO: 309 53G1 NTNSRHSGVPSRFSGSILGNKAALTITGAQADDESDYFC ALFISNPSVE FGGGTQLTVLG SEQ ID NO: 110SEQ ID NO: 298 SEQ ID NO: 160 SEQ ID NO: 309 35G3 NTNSRHSGVPDRFSGSILGNKAALTIRGAQADDEAEYYC ALFISNPSVE FGGGTQLTVLG SEQ ID NO: 110SEQ ID NO: 299 SEQ ID NO: 160 SEQ ID NO: 309 53C1 NTNSRHSGVPDRFSGSILGNKAALTIRGAQADDEAEYYC ALFISNPSVE FGGGTQLTVLG SEQ ID NO: 110SEQ ID NO: 299 SEQ ID NO: 160 SEQ ID NO: 309 35F6 NTNSRHSGVPDRFSGSILGNKAALTITGAQPEDESDYFC ALFISNPSVE FGGGTQLTVLG SEQ ID NO: 110SEQ ID NO: 300 SEQ ID NO: 160 SEQ ID NO: 309 36G2 NTNTRHSGVPDRFSGSILGNKAALTITGAQPEDESDYYC ALFISNPSVE FGGGTQLTVLG SEQ ID NO: 119SEQ ID NO: 301 SEQ ID NO: 160 SEQ ID NO: 309 39D5 NTNTRHSGVPDRFSGSILGNKAALTITGAQPEDESDYYC ALFISNPSVE FGGGTQLTVLG SEQ ID NO: 119SEQ ID NO: 301 SEQ ID NO: 160 SEQ ID NO: 309 42D12 NTNTRHSGVPSRFSGSILGNKAALTITGAQADEDEAEYFC ALFISNPSVE FGGGTQLTVLG SEQ ID NO: 119SEQ ID NO: 302 SEQ ID NO: 160 SEQ ID NO: 309 35G1 NTNSRHSGVPDRFSGSILGNKAALTITGAQADDESDYFC ALFISNPSVE FGGGTQLTVLG SEQ ID NO: 110SEQ ID NO: 297 SEQ ID NO: 160 SEQ ID NO: 309 41D12 NTNTRHSGVPDRESGSILGNKAALTITGAQADDEAEYFC ALFISNPSVE FGGGTQLTVLG SEQ ID NO: 119SEQ ID NO: 303 SEQ ID NO: 160 SEQ ID NO: 309 41H8 NTNSRHSGVPDRFSGSILGNKAALTITGAQPEDESDYFC ALFISNPSVE FGGGTQLTVLG SEQ ID NO: 110SEQ ID NO: 300 SEQ ID NO: 160 SEQ ID NO: 309 35G2 NTNSRHSGVPDRFSGSILGNKAALTITGAQADDEADYFC ALFISNPSVE FGGGTQLTVLG SEQ ID NO: 110SEQ ID NO: 304 SEQ ID NO: 160 SEQ ID NO: 309 40F1 NTNTRHSGVPDRFSGSILGNKAALTITGAQADDEAEYFC ALFISNPSVE FGGGTQLTVLG SEQ ID NO: 119SEQ ID NO: 303 SEQ ID NO: 160 SEQ ID NO: 309 53B1 NTNTRHSGVPDRFSGSILGNKAALTITGAQADDEAFYFC ALFISNPSVE FGGGTQLTVLG SEQ ID NO: 119SEQ ID NO: 303 SEQ ID NO: 160 SEQ ID NO: 309 39C3 NTNTRHSGVPSRFSGSILGNKAALTITGAQADDESDYYC ALFISNPSVE FGGGTQLTVLG SEQ ID NO: 119SEQ ID NO: 305 SEQ ID NO: 160 SEQ ID NO: 309 53E1 NTNTRHSGVPSRFSGSILGNKAALTITGAQADDESDYYC ALFISNPSVE FGGGTQLTVLG SEQ ID NO: 119SEQ ID NO: 305 SEQ ID NO: 160 SEQ ID NO: 309 53H1 NTNTRHSGVPDRFSGSILGNKAALTITGAQADDEAEYFC ALFISNPSVE FGGGTQLTVLG SEQ ID NO: 119SEQ ID NO: 303 SEQ ID NO: 160 SEQ ID NO: 309 53A2 NTNTRHSGVPDRFSGSILGNKAALTITGAQADDEAEYFC ALFISNPSVE FGGGTQLTVLG SEQ ID NO: 119SEQ ID NO: 303 SEQ ID NO: 160 SEQ ID NO: 309

TABLE 15BVL amino acid sequences of germlined 27B3 clones with more than94% total human identity, with sequence identifiers for the fullVL sequence. Humanizing mutations are italicized and primer-introduced mutations are bolded. SEQ LIGHT ID CHAIN NO: CLONEFULL VL SEQUENCE 201 27B3QAVVTQEPSLTVSPGGTVTLTCGLKSGSVTSTNEPTWYQQ7PGQAPRLLIYNTNTRHSGVPSRFSGSISENKAALTITGAQPFDEAEYFCALFISNPSVEFGGGTQLTVLS 230 36A9QTVVTQEPSFSVSPGGTVTLTCGLKSGSVTSDNFPTWYQQTPGQAPRLLIYNTNTRHSGVPDRFSGSILGNKAALTITGAQADDESDYFCALFISNPSVEFGGGTQLTVLG 231 53F1QAVVTQFPSFSVSPGGTVTLTCGLKSGSVTSDNFPTWYQQTPGQAPRLLTYNTNTRHSGVPDRFSGSILGNKAALTITGAQADDESDYFCALFISNPSVEFGGGTQLTVLG 232 36D6QTVVTQEPSLTVSPGGTVTLTCGLKSGSVTSTNFPTWYQQTPGQAPRLLIYNTNSRHSGVPDRFSGSILGNKAALTITGAQADDFSDYFCALFISNPSVFFGGGTQLTVLG 233 53G1QTVVTQEPSFSVSPGGTVTLTCGLTSGSVTSTNFPTWYQQTPGQAPRLLIYNTNSRHSGVPSRFSGSILGNKAALTITGAQA0DESDYFCALFISNPSVEFGGGTQLTVLG 234 35G3QAVVTQEPSFSVSPGGTVTLTCGLKSGSVTSDNFPTWYQQTPGQAPRLLIYNTNSRHSGVPDRFSGSILGNKAALTIRGAQADDEAEYYCALFISNPSVEFGGGTQLTVLG 235 53C1QTVVTQEPSFSVSPGGTVTLTCGLKSGSVTSDNFPTWYQQTPGQAPRLLIYNTNSRHSGVPDRFSGSILGNKAALTIRGAQADDEAEYYCALFISNPSVEFGGGTQLTVLG 236 35F6QTVVTQEPSFSVSPGGTVTLTCGLTSGSVTSDNFPTWYQQTPGQAPRLLIYNTNSRHSGVPDRFSGSILGNKAALTITGAQPEDESDYFCALFISNPSVEFGGGTQLTVLG 237 36G2QAVVTQEPSLIVSPGGTVTLTCGLKSGSVTSDNFPTWYQQTPGQAPRLLIYNTNTRHSGVPDRFSGSILGNKAALTITGAQPEDESDYYCALFISNPSVEFGGGTQLTVLG 238 39D5QTVVTQEPSLTVSPGGTVTLTCGV TKSGSVTSDNFPTWYQQTPGQAPRLLIYNTNTRHSGVPDRFSGSILGNKAALTITGAQPEDESDYYCALFISNPSVEFGGGTQLTVLG 239 42D12QAVVTQEPSFSVSPGGTVTLTCGLTSGSVTSTNFPTWYQQTPGQAPRLLIYNTNTRHSGVPSRFSGSILGNKAALTITGAQADDEAEYFCALFISNPSVEFGGGTQLTVLG 240 35G1QTVVTQEPSFSVSPGGTVTLTCGLKSGSVTSDNFPTWYQQTPGQAPRLLIYNTNSRHSGVPDRFSGSILGNKAALTITGAQADDESDYFCALFISNPSVEFGGGTQLTVLG 241 41D12QAVVTQEPSLIVSPGGTVTLTCGLKSGSVTSDNFPTWYQQTPGQAPRLLIYNTNTRHSGVPDRFSGSILGNKAALTITGAQADDFAEYECALFISNPSVEFGGGTQLTVLG 242 41H8QAVVTQEPSFSVSPGGTVTLTCGLTSGSVTSTNFPTWYQQTPGQAPRLLIYNTNSRHSGVPDRFSGSILGNKAALTITGAQPEDESDYFCALFISNPSVEFGGGTQLTVLG 243 35G2QTVVTQEPSFSVSPGGTVTLTCGLKSGSVTSTNFPTWYQQTPGQAPRLLIYNTNSRHSGVFDRFSGSILGNKAALTITGAQADDEADYFCALFISNPSVEFGGGTQLTVLG 244 40F1QAVVTQEPSFSVSPGGTVTLTCGLKSGSVTSDNFPTWYQQTPGQAPRLLIYNTNTRHSGVPDRFSGSILGNKAALTITGAQADDEAEYFCALFISNPSVEFGGGTQLTVLG 245 5381QTVVTQEPSFSVSPGGTVTLTCGLKSGSVTSDNFPTWYQQTPGQAPRLLIYNTNTRHSGVPDRFSGSILGNKAALTITGAQADDEAEYFCALFISNPSVEFGGGTQLTVLG 246 39C3QAVVTQEPSFSVSPGGTVTLTCGLKSGSVTSDNFPTWYQQTPGQAPRLLIYNTNTRHSGVPSRFSGSILGNKAALTITGAQADDESDYYCALFISNESVEFGGGTQLTVLG 247 53E1QTVVTQEPSFSVSPGGTVTLTCGLKSGSVTSDNEPTWYQQTPGQAPRLLIYNTNTRHSGVPSRFSGSILGNKAALTITGAQADDESDYYCALFISNPSVEFGGGTQLTVLG 244 53H1QTVVTQEPSFSVSPGGTVTLTCGLKSGSVTSDNFPTWYQQTPGQAPRLLTYNTNTRHSGVPDRFSGSILGNKAALTITGAQADDEAEYECALFISNPSVEEGGGTQLTVLG 245 53A2QAVVTQEPSFSVSPGGTVTLTCGLKSGSVTSDNEPTWYQQTPGQAPRLLIYNTNTRHSGVPDRFSGSILGNKAALTITGAQADDEAEYFCALFISNPSVEFGGGTQLTVLG

Example 15: Temperature Stability of Germlined 27B3 mAbs

Several germlined 27B3 mAb variants were expressed in HEK293 cells asfull-length human IgG1 molecules. After protein A purification, thetemperature stability of the germlined 27B3 mAb clones was assessed.Specifically, each mAb was incubated at various temperatures (2 or 5° C.intervals apart) for 1 hour at a concentration of 100 μg/ml in PBSbuffer. The temperature of the mAbs was then decreased in a controlledmanner (reduction to 25° C. over 2 hours, followed by overnightincubation at 4° C.) and centrifugation was used to remove aggregates.The amount of active mAb (as percentage of the total mAb concentration)in the supernatant was measured by Biacore.

The results (set forth in Table 17) demonstrate that most clones have asimilar melting temperature (T_(m)) as wild type 27B3 has. Surprisingly,clones 41D12 and 35G2 show to have an increased thermostability ascompared to 27B3.

TABLE 17 Temperature stability of germlined 27B3 mAbs mAb Clone T_(m) [°C.] 27B3 (WT) 64.7 64.6 35G1 63.7 35G3 61.7 53A2 62.2 62.2 41D12 65.965.9 35G2 65.6 66.0 40F1 63.5 53H1 64.1The temperature stability of germlined 27B3 mAbs: 41D12, 35G2 and 40F1was further studied using a variety of techniques. Aliquots (1 ml) ofthe test mAbs at a concentration of 100 μg/ml were prepared inDelbucco's PBS containing 0.02% Tween. A negative control aliquotconsisting of buffer only (Delbucco's PBS containing 0.02% Tween) and apositive control aliquot consisting of mAb in Delbucco's PBS containing0.02% Tween were also prepared. For each mAb preparation, aliquots werestored at 4° C., at RT and at 37° C. for a period of 0-8 weeks. 50 μlsamples were removed from the aliquots on days 1, 7, 14, 21, 28, 35, 56and 92 for analysis. A sample was stored at −20° C. and used asreference.15.1 Gelfiltration Analysis of Samples

Samples taken at each time point were also analysed by gelfiltrationusing a Superdex200 10/300 GL column. The 50 μl test sample was added to200 μl PBS+0.02% Tween-20. A sample (125 μl) was taken for gelfiltrationanalysis. Samples were centrifuged before analysis to remove largeraggregates. The % of monomeric peak and area of monomeric peak wasmeasured, as well as the retention volume. The results are shown inTables 19, 20 and 21.

TABLE 19 Results of PBS stability study - % monomeric peak d d d d d d dd 1 7 14 21 28 35 56 92 35G2 Ref- 99.82 99.84 99.81 99.81 99.80 99.8740F1 er- 99.85 99.83 99.83 99.83 99.87 99.86 41D12 ence 99.84 99.8199.84 99.75 99.73 99.23 99.82 99.84 35G2 4° C. 99.85 99.75 99.74 99.8399.66 99.84 40F1 99.65 99.29 99.73 99.77 99.72 41D12 99.88 99.76 99.8399.8 99.84 99.81 99.49 35G2 RT 99.78 99.83 99.81 99.87 99.83 40F1 99.8799.83 99.84 99.77 99.77 41D12 99.88 99.86 99.87 99.78 99.73 99.80 99.7135G2 37° 99.86 99.80 99.72 99.68 99.48 99.32 40F1 C. 99.83 99.78 99.7899.77 99.40 99.27 41D12 99.89 99.77 99.61 99.71 99.45 99.02 98.66 97.46

TABLE 20 Results of PBS stability study - area under curve of themonomeric peak (mAU*ml) d d d d d d d d 1 7 14 21 28 35 56 92 35G2 Ref-67.34 68.46 64.92 66.70 66.69 66.37 40F1 er- 67.04 67.96 70.36 69.6269.53 67.23 41D12 ence 66.41 67.64 61.17 68.53 68.60 66.08 67.48 67.0635G2 4° C. 68.41 67.58 62.55 68.64 68.12 68.42 40F1 69.86 67.31 65.5566.76 68.99 41D12 68.38 69.87 65.61 68.98 69.99 68.85 67.33 35G2 RT66.76 67.23 71.40 68.55 68.45 40F1 68.42 69.53 69.17 68.02 70.32 41D1266.47 69.01 64.32 69.72 71.56 69.36 67.16 35G2 37° 67.52 67.14 64.4961.89 66.51 65.64 40F1 C. 66.48 67.69 67.35 68.02 68.61 67.23 41D1267.09 68.13 61.51 68.97 68.37 68.25 67.78 66.51

TABLE 21 Results of PBS stability study - retention volume (ml) d d d dd d d d 1 7 14 21 28 35 56 92 35G2 Ref- 12.17 12.13 12.13 12.15 12.1712.20 40F1 er- 12.14 12.09 12.11 12.12 12.14 12.16 41D12 ence 12.1212.08 12.10 12.10 12.12 12.14 12.10 12.13 35G2 4° C. 12.16 12.12 12.1412.16 12.17 12.20 40F1 12.13 12.09 12.11 12.12 12.14 41D12 12.12 12.0812.10 12.10 12.13 12.10 12.13 35G2 RT 12.16 12.12 12.14 12.16 12.17 40F112.14 12.09 12.10 12.12 12.13 41D12 12.12 12.07 12.10 12.10 12.28 12.0912.13 35G2 37° 12.16 12.12 12.13 12.14 12.16 12.17 40F1 C. 12.13 12.0912.09 12.12 12.12 12.14 41D12 12.12 12.08 12.08 12.09 12.10 12.12 12.0612.08The results of gelfiltration analysis of 35G2, 40F1 and 41D12 samplestaken after 5 weeks incubation at 37° C. are shown in FIG. 13. Two minorpeaks are visible at a higher retention time (indicating the presence ofsome aggregates) and lower retention time (indicating the presence ofsome degradation product). The results demonstrate that the majority ofthe protein is intact and does not aggregate.Analysis of Samples by BIACORE

Samples taken at certain time points were tested for potency in CD70binding using Biacore. The 50 μl sample was added to 200 μl PBS+0.02%Tween, and diluted 1/400 as follows: 5 μl of sample (1 mg/ml)+195 μlHBS-EP+(Biacore buffer), further diluted 10 μl+90 μl HVS-EP+=2.5 μg/ml.Biacore analysis was performed using a highly CD70 coated CM5 chip (4000RU). The results for mAbs 40F1, 35G2 and 41D12 are shown in FIG. 16. Thereference sample is the 100% sample.

The results demonstrate that the mAbs loose some potency (affinity forthe antigen CD70) over time when incubated at 37° C. For 41D12, both theslope and the maximal signal (R0) are plotted (see FIGS. 16C and D).

Next, the freeze-thaw stability of germlined 27B3 mAbs: 41D12, 35G2 and40F1 was studied. Therefore, a 0.7 ml aliquot of the mAbs (at 5 mg/ml)was frozen for at least 6 hours at −20° C. and thawed for 1 hour at RT.This cycle was repeated 9× (so 10 freeze-thaw cycles in total) andsamples were analyzed by gelfiltration as above. The results demonstratethat the mAbs are stable upon freeze thawing for 10 cycles: nodegradation products or aggregates were observed in gelfiltration.

TABLE 22 Results of gelfiltration stability study PBS (left = valueafter 10 F/T cycles, right is value for reference sample) 35G2 40F141D12 Gel Filtration 10/ref 10/ref 10/ref % monomeric peak 99.9/99.899.8/99.7 99.8/99.8 Area monomeric peak 67.5/64.7 65.9/67.2 68.2/67.8Retention time 12.20/12.21 12.15/12.15 12.12/12.12Samples were also tested for potency in Biacore as described above. Theresults demonstrate that after 10 freeze-thaw cycles the mAb still has97% of its potency (for all three mAbs tested.

TABLE 23 Potency in Biacore upon 10 freeze-thaw cycles (%) 35G2 40F141D12 Reference 100 100 100 10xFT 97.35 96.96 97.02

Example 16: ADCC Potency of Germlined 27B3 mAbs

Several germlined 27B3 variants were expressed in HEK293 cells asfull-length human IgG1 molecules. After protein A purification, the ADCCpotency of the germlined 27B3 mAbs was assessed. ADCC was measured usingas described above in example 7.

The relative IC50's for the germlined 27B3 mAbs compared to the parental27B3 are given in table 18 for both ADCC potency and CD70/CD27 blockingpotency in the Raji co-culture assay (as described in example 6). Afigure of <1.0 denotes improved IC50 for germlined variant relative toparental 27B3. From these data it can be concluded that germlinedvariant 41D12 maintained the ADCC potency as well as the CD70/CD27blocking potency combined with the best T_(m).

TABLE 18 Relative IC50's of germlined 27B3 variants in the 786-O basedADCC assay and neutralization in Raji based bioassay compared to that ofparental mAb clone Raji co-culture mAb assay IC50 ADCC IC50 of germlinedclone relative to 27B3 mAb relative to 27B3 27B3 1.0 1.0 1.0 1.0 1.0 1.01.0 53A2 0.8 1.5 1.9 1.5 4.6 0.7 41D12 1.2 1.7 1.1 2.2 1.4 2.0 2.0 35G21.4 1.7 2.1 4.4 1.4 1.9 2.3 40F1 1.8 1.0 1.5 1.3 1.5 2.1 1.6 53H1 1.60.9 1.8 3.4 2.1 1.9 1.7

Example 17: Construction and Selection of Cell Lines Expressing theNon-Fucosylated CD70 mAb, ARGX-110

A double gene vector encoding the VH and VL amino acid sequences ofgermlined 27B3 clone 41D12 was produced (za allotype). Nucleotidesequences encoding the heavy chain (SEQ ID NO:344) and light chain (SEQID NO:345) of 41D12 are given in Table 30 below.

Potelligent® CHOK1SV cells were stably transfected with the double genevector by electroporation. Six rounds of transfection were performed. Ineach round, the cells from each electroporation were added to 200 mL ofchemically defined animal component free (CDACF) medium CM63 and platedout over forty 96-well plates at 50 μL per well. The day aftertransfection, 150 μL of the CM63 medium containing the selective agentL-methionine sulphoximine (MSX) was added to each well to give a finalMSX concentration of 50 pM. After approximately 3, 4 and 5 weeks ofincubation the plates were screened for the presence of colonies using acloning mirror. Any colonies identified were examined further using amicroscope to evaluate if the colony had arisen from a single ormultiple cells.

Two hundred and forty-five colonies were identified and screened forantibody production using an Octet based method. Antibody concentrationsranged from 0 to 172 μg/mL. Of the 245 cell lines screened, 214 werepositive for antibody production. The cell lines were ranked based onproductivity and the top 70 cell lines were selected. Cultures of these70 cell lines were initially expanded in static culture and subsequentlyinto suspension culture. Thirty cell lines that exhibited acceptablegrowth were selected and evaluated further in a batch shake-flaskculture productivity screen. Cultures were gassed on days 4, 6, 8 and 10and harvested on day 12. The concentration of antibody in the harvestsupernatant samples was determined in HPLC using Protein A containingcolumn. The antibody concentrations in the samples from the 30 celllines ranged from 109 to 877 mg/L. The results of the batch shake-flaskassessment were used to rank the cell lines, based on productivity. Thetop 20 cell lines were selected for further evaluation.

The growth and productivity data for these 20 selected cell linesexpressing the ARGX-110 antibody was studied in fed-batch shake-flaskcultures using CDACF medium and the results are shown in Table 24.Antibody concentrations at harvest ranged from 857 to 3922 mg/L, asdetermined on a Protein A HPLC column. Cell line F13 was selected toinoculate a disposable 10 L cell bag bioreactor. A harvest antibodyconcentration of 4327 mg/L was achieved. This cell line achieved anantibody concentration at harvest of 2711 mg/L in fed-batch shake-flaskculture and had the second highest specific production rate (2.22pg/cell/h) of all 6 cell lines assessed (B1, D4, D5, F1, F13 and F18).This cell line also exhibited acceptable growth characteristics.

TABLE 24 Summary of growth and productivity data for 20 selected celllines, expressing the ARGX-110 antibody, grown in CDACF fed-batchshake-flask cultures. Maximum Viability Antibody Specific Viable CellIVC at at Concentration Production Concentration Harvest⁽¹⁾ Harvest atHarvest⁽²⁾ Rate (q_(P))⁽³⁾ Cell Line (10⁶/mL) (10⁹ cell · h/L) (%)(mg/L) (pg/cell/h) A1 8.55 1276 47.1 1536 1.204 A9 4.01 1039 71.5 10330.994 B1 8.60 1777 69.9 3922 2.206 B2 5.22 1277 59.7 2938 2.301 D4 9.101957 79.5 3346 1.709 D5 7.84 1791 78.5 3258 1.819 D12 7.76 1635 91.21424 0.871 D20 7.66 1525 60.6 3130 2.052 D24 4.25 973 28.3 1133 1.164D30 7.58 1492 93.4 2095 1.405 F1 8.55 1717 41.0 3704 2.157 F13 6.14 122446.0 2711 2.215 F18 7.80 1641 50.0 3301 2.011 F20 7.76 1812 68.2 20951.156 F21 7.95 1693 39.7 2008 1.186 F22 8.13 1953 87.9 1572 0.805 F299.16 1944 88.3 1435 0.738 F31 7.27 1453 73.9 1540 1.060 F33 5.67 14097.2 857 0.608 F34 10.04 2049 93.4 1328 0.648 ⁽¹⁾Time integral of theviable cell concentration at harvest. ⁽²⁾Determined by Protein A HPLC.⁽³⁾Calculated by linear regression analysis of the antibodyconcentration against the time integral of the viable cellconcentration.

Example 18: Affinity of CD70 mAbs

18.1 Affinity for Cancer Cell Lines Expressing CD70

Several cancer cell lines were tested by FACS analysis for binding ofARGX-110 and SGN70 (described in US2010/0129362) at saturating mAbconcentrations (at least 2 μg/ml). This was either done at 4° C. (1 hourincubation of cells with mAbs) or at 37° C. (15 minutes incubation ofcells with mAbs). Binding was detected using anti-hIgG1-Fc-FITC (AF006,Binding Site) or anti-hIgG1-Fc-PE antibody (eBioscience, 12-4998).Fluorescence was measured using a flow cytometer. The results aresummarized in Table 25. The third column of the table shows whetherARGX-110 binds with low (+), medium (++) or high (+++) affinity to thevarious cell lines tested.

In the right-hand column, the FACS signal (MFI) for ARGX-110 is dividedby the FACS signal (MFI) for SGN70 for the experiment carried out at 37°C. These results demonstrate that ARGX-110 binds with higher affinity tothe cells than SGN70, particularly for lower copy-number cell lineswhere it can be expected that high affinity binding of an antibody canbe picked up more easily.

TABLE 25 Binding of CD70 mAbs ARGX-110 and SGN70 to cancer call linesSignal in FACS of ARGX- 110/signal in FACS Type Cell line ARGX-110 ofSGN70 Burkitt lymphoma Raji +++ 1.2 Large B cell lymphoma SU-DHL-6 + 2.4Hodgkin lymphoma L428 +++ 1.4 Non Hodgkin lymphoma MHHPREB1 +++ 1.2Mantle Cell Lymphoma Mino +++ 1.0 Jeko +++ Granta 519 ++ 0.9 Rec-1 +Chronic lymphocytic leukemia Mec1 +++ 1.2 JVM-3 ++ JVM-2 + 2.4 CutaneousT cell lymphoma HUT78 +++ 1.0 HH + 1.7 Multiple Myeloma U266 +++ 0.8JNN-3 +++ LP1 ++ AMO-1 +++ RPMI8226 +++ MM1.S ++ KMS11 +++ KMS12MB +++Renal cell carcinoma 786-O +++ 2.1 Caki-1 ++ 1.9 A498 ++ 1.0 AstrocytomaU251 +++ Gastric carcinoma MKN-45 + 10.6 Lung carcinoma A549 + 3.8EBC-1 + 8.9 Melanoma WM1205-Lu +++ WM852 + WM3248 +++ WM793 + WM1552C ++WM115 +++ Glioblastoma GaMG + U87MG ++ 2.0 U343 ++ Ovarian carcinomaOAW-42 + SKOV3 + OVCAR3 + Pancreatic carcinoma PANC-1 ++ 2.9 PANC-89 +

In a further experiment, the apparent binding affinity of CD70 mAbs,ARGX-110, SGN70 and MDX1411 [see above], across a range ofconcentrations was determined for different cell lines. Cells wereincubated for 1 hour at 4° C. with a dilution series of CD70 bindingmAbs and binding was detected using anti-hIgG1-Fc-FITC oranti-hIgG-Fc-PE antibody. Fluorescence was measured using a flowcytometer and the median fluorescence was plotted. The results are shownin FIG. 15.

The results demonstrate that the affinity of the three different mAbsfor CD70 on cells is comparable, but for some cell lines, SU-DHL-6, A549and MKN45, binding of ARGX-110 is superior as compared to MDX1411 andSGN70. The EC50 for binding to SU-DHL-6, A549 and MKN45 is much higherfor all mAbs as compared to EC50 on the other cell lines like U266 andmany others, which probably indicates that the mAb is binding only withone arm (Fab), no longer allowing for avidity and thus resulting inlower affinities. Indeed, when tested in Biacore, the affinity of theFab of ARGX-110 versus SGN70 and MDX1411 is much higher (see below).

18.2 Affinity for Patient Cells Expressing CD70

Primary cells taken from chronic lymphocytic leukaemia (CLL) patients (2high risk patients, 1 low risk patient) were plated at 250,000cells/well in round bottom 96 well plates in RPMI 1640+10% FBS and mAbsat a concentration of 5 μg/ml in RPMI+10% FBS were added. Afterincubation for up to 5 hours at 37° C., cells were washed twice withice-cold PBS. Alexa Fluor 647 labelled goat Anti-Human IgG (InvitrogenCat #21445) was diluted 1/500 in PBS/1% BSA and incubated for 20 minutesat 4° C. The plate was washed twice with ice-cold PBS and 4%paraformaldehyde was added for 15 minutes at room temperature to fix thecells. Signals were measured by FACS. The results are shown in FIG. 16.The results demonstrate that ARGX-110 binds well to the cells from theCLL patients whereas SGN70 almost gave no binding signals.

18.3 Affinity of CD70 Fabs for recombinant CD70 in Biacore

Recombinant human CD70 was immobilized on a CM5 Biacore chip. Theimmobilization was performed in accordance with a method provided byBiacore and by using the NHS/EDC kit (Biacore AB): after activation ofthe chip, a solution of 50 μg/ml of recombinant CD70 in 10 mM acetatebuffer with pH of 5 was prepared and 1 μl of this solution (50 ng) wasinjected resulting in a surface density of approximately 1000 RU.

Fabs were prepared for the CD70 mAbs ARGX-110, MDX1411 and SGN70 bypapain digestion. These Fabs, at a concentration of approximately100-400 μg/ml, were diluted 6-fold in HEPES-buffered saline (0.1M HEPES,1.5M NaCl, 30 mM EDTA, 0.5% v/v surfactant P20). They were injected (30μl) and passed through the flow cells at a flow rate of 30 μl/min. Afterbinding of the Fab to CD70, the off-rate was monitored for a period of10 minutes. After dissociation, the flow cell surfaces were regeneratedby injecting 5 μl of 10 mM NaOH. Sometimes multiple injections of NaOHwere needed to regenerate the surfaces depending on the affinity of theFabs. Off-rate analysis was done by applying the BIAevaluation software.First, the sensogram of the blank runs were subtracted from thoseobtained with the coated flow cell. Then the off-rate was determined fora time range of 10 minutes using the Fit kinetics application and the Kdvalue was calculated. The off rates are summarized in Table 26.

TABLE 26 “off rate” of Fabs for binding to human CD70 Off rate [10⁻⁴s⁻¹] ARGX-110 1.4 SGN70 7 MDX1411 1718.4 Spiking Experiments to Assess Lysis of SU-DHL-6 Cells Bound by CD70mAbs

SU-DHL-6 cells (a diffuse large B cell lymphoma cell line) were pipetedinto wells at 50,000 cells/well which were spiked then into 300,000freshly isolated PBMCs/well from healthy donors and mAbs were added atdifferent concentrations. The samples were incubated for 2 days andanalysed by FACS for the depletion of the cell line (as measured withanti-human CD19 APC (eBioscience 17-0199-42)). The best potency in cellkilling was obtained with ARGX-110, which also gives the highest % oflysis. The results are shown in FIG. 17.

Example 19: CD27-CD70 Blocking Activity of CD70 mAbs

The interaction between CD70 and CD27 may contribute to tumour cellsurvival, proliferation and/or immune suppression within the tumourmicroenvironment. The ability of CD70 mAbs to block the interactionbetween CD70 and CD27 was therefore assessed by ELISA.

In this assay, a microtiter plate (Nunc Maxisorb) was first coated with100 μl anti-FLAG M2 monoclonal antibody (Sigma Aldrich, F3165) at1250-fold dilution in PBS (to achieve a final concentration ofapproximately 3.5 μg/ml) overnight at 4° C. The plate was washed oncewith PBS-Tween and incubated at RT for 2 hours with 300 μl PBS-1%casein. The plate was washed a further three times with PBS-Tween. 100μl of 5 ng/ml (80 pM) Flag-TNC-CD70 (The Journal of Immunology, 2009,183: 1851-1861) diluted in PBS-0.1% casein was added to the plate andincubated at RT for 1 hour while shaking. The plate was washed fivetimes with PBS-Tween. The CD70 mAb to be tested was added to the plate;various concentrations were achieved by diluting the stock antibodysolution in PBS-0.1% casein. Immediately thereafter, 50 μl of 1 μg/ml(final concentration 6.5 nM) recombinant human CD27 Fc chimera (R&Dsystems 382-CD, MW=46.5 kDa) was added, and incubated at RT for 1 hourwhile shaking. The plate was washed five times with PBS Tween. 100 μl ofbiotinylated anti-CD27 (eBioscience 13-0271), diluted 500-fold inPBS-0.1% casein, was added, and the plate incubated at RT for a furtherhour, while shaking. The plate was washed five times with PBS Tween. 100μl of Strep-HRP (Jackson Immunoresearch 016-030-084) diluted 5000 foldin PBS-0.1% casein was added, and the plate incubated at RT for afurther hour, while shaking. The plate was washed five times withPBS-Tween. 100 μl TMB was added to the plate and the OD at 620 nm wasmeasured.

Using this assay, the blocking potency of ARGX-110 was compared to thepotency of SGN70 and MDX1411. As shown in FIG. 18, ARGX-110 is much morepotent (about 100-fold) in blocking the interaction between CD70 andCD27 than the two benchmark mAbs (IC50 ARGX-110=67 ng/ml, MDX1411=5500ng/ml and SGN70=4972 ng/ml).

Example 20: Binding Properties of CD70 mAbs

20.1 Cross-Reactivity with CD70 of Non-Human Species

Determining the animal cross-reactivity of CD70 mAbs is useful for thepurposes of assessing which animal models can be used for in vivo proofof concept studies, and which species are most suited for toxicologystudies. Rhesus CD70 is 94% identical to human CD70 and Cynomolgusmonkey CD70 is 95% identical to human CD70. An alignment of thesequences from the different species is shown in FIG. 19. The mouse andrat CD70 sequences are also included so as to highlight the differencesas compared with the human sequence.

CD70 mAbs ARGX-110, SGN70 and MDX1411 were tested for binding to U266 (ahuman multiple myeloma cell line), LCL8664 cells (rhesus B cell lymphomacell line, ATCC-CRL-1805) and to HSC-F cells (cynomolgus monkey T-cellline, fetal spleen-derived lymphocytes, Japanese health sciencefoundation) cells in a dilution series starting at a concentration of10-20 μg/ml.

Detection was performed using goat anti human IgG1 FITC. Samples wereanalysed by FACS. The results are shown in FIG. 20. The copy-number ofCD70 on both monkey cell lines is very low (low signals in FACS).However, ARGX-110 has a high affinity for cynomolgus and rhesus CD70 ascompared to the affinity of SGN70 and MDX1411 for CD70 of these species.

20.2 Blocking Potency of CD70 mAbs

The ability of CD70 mAbs (ARGX-110, SGN70 and MDX1411) to block theinteraction between CD70 and CD27 was tested using the blocking ELISAdescribed in Example 19 above. The assay was adapted such to measure theblocking potency of each antibody for human, rhesus and cynomolgusmonkey Flag-TNC-CD70 (Wyzgol, et al., J. Immunol., 2009, 183:1851-1861).

In the ELISA, exactly the same conditions were used for CD70 from thedifferent species so that the IC50 values between the different speciescould be compared directly. The results are shown in FIG. 21 and supportthe results seen using cells from humans, rhesus monkeys and cynomolgusmonkeys. ARGX-110 has an unaltered potency and therefore an unalteredaffinity for CD70 of monkeys as compared to human, whereas SGN70 andespecially MDX1411 have a lower affinity for monkey than for human CD70.IC50 values are summarized in Table 27.

TABLE 27 IC50 for blocking of the CD27-CD70 interaction in ELISA forCD70 from different species IC50 [ng/ml] in inhibition ELISA RhesusCynomolgus Cells Human monkey monkey ARGX-110 39 75 48 SGN70 54 178 112MDX1411 44 >10000 >1000020.3 Binding to Denatured CD70

The binding of CD70 mAbs to denatured recombinant CD70 was assessed byELISA. Recombinant CD70 was denatured by heating for 5 minutes at 95°C., followed by immediate cooling on ice for 5 minutes. A microtiterplate was coated with 5 μg/ml CD70 with or without denaturation. Theplate was blocked and the CD70 mAbs for testing were applied at 10μg/ml. After washing, binding of the mAbs was detected using anti-humanIgG-HRP. As a positive control for the coating, instead of using theCD70 mAbs, an anti-Flag mouse-derived mAb was applied and detection waswith anti-mouse-HRP. TMB was used as a substrate and OD at 620 nm wasmeasured. The results from this ELISA are shown in FIG. 22. The resultsdemonstrate that ARGX-110 binds to a different epitope than SGN70,MDX1411 (MDX2H5) and MDX69A7 as ARGX-110 still binds to denatured CD70whereas the other mAbs don't. Amongst the other mAbs, some are alsobinding to denatured CD70 (fe 5F4, 9G2, 57B6).

20.4 Epitope Mapping Using Mouse-Human Chimeras

Human-mouse CD70 ECD fusion proteins were constructed by exchangingdomains of human and mouse CD70 in order to map the domain recognitionof the mAbs. The construction was done using standard recombinant DNAand PCR methodologies. The mouse and human chimeras were cloned into aeukaryote expression vector with a Flag tag for capturing in ELISA and aTNC for trimerisation of the protein (Flag-TNC-CD70). Proteins wereexpressed as soluble proteins in HEK293 cells. The sequence of thedifferent chimeras is shown in FIG. 23.

A Maxisorb (Nunc) microtiter plate was coated with 3.5 μg/ml mouse M2anti-Flag mAb (Sigma Aldrich, F3165) and the different chimeras werecaptured. A dilution series of the CD70 mAbs to be tested was appliedstarting at a concentration of 10 μg/ml and making 3-fold dilutions.After 2 hours, binding was detected using anti-human-Fc-HRP at a16000-fold dilution. Staining was done with ABTS and OD at 405 nm wasmeasured. As a positive control, CD27-Fc was applied which is able tobind to both human and mouse CD70 with good affinity as well as to thechimeric human-mouse CD70 variants. The EC50 values for binding aresummarized in Table 28.

TABLE 28 Binding of ARGX-110 to human-mouse CD70 chimeras shown in FIG.23. EC50 [ng/ml] ARGX-110 CD27 human 22 79 chim-1 27 159 chim-2.1 15 51chim-2.2 16 56 chim-2.3 15 chim-2.4 14 76 chim-2 NB 99 chim-3 NB 94chim-4 NB 51 mouse NB 41 NB = no binding

The results demonstrate that ARGX-110 can bind to human Flag-TNC-CD70and to chimeras 1, 2.1, 2.2, 2.3 and 2.4 but does not bind to chimeras2, 3 and 4 and mouse Flag-TNC-CD70. This indicates that the epitope forARGX-110 is within the following amino acid sequence: HIQVTLAICSS (SEQID NO:342).

Example 21: CD70 Internalization of Different Tumor Cell Lines andPrimary Tumor Cells

It has been demonstrated that binding of CD70 antibodies to the renalcarcinoma derived cell lines 786-O and A498 results in the rapid (within1 hour) internalization of the antibody-receptor complex (Adam et al.,Br. J. Cancer (2006) 95: 298-306). In order to test for internalizationof mAbs ARGX-110, SGN70 and MDX1411, 786-O cells were cultured in a96-well microtiter plate and incubated overnight at 37° C. 2.5 μg/ml mAbwas added and incubated with the cells for 0-24 hours at 37° C. Plateswere washed 3 times 5′ with stripping buffer (150 mM NaCl, 100 mMGlycine, pH=2.5; coded “IN” representing the amount of mAb internalizedvia CD70) or PBS (coded “OUT” and representing the amount of mAb boundto the receptor at the outside of the cell). Subsequently, cells werefixed with 4% paraformaldehyde for 30′ at RT, washed with PBS, andincubated 5′ with 0.2% Triton-X-100 (“IN”) or PBS (“OUT”). Next, cellswere washed twice and incubated at RT for 10′ with 100 mM glycinefollowed by 30 mins incubation with PBS+1% BSA. Finally cells werestained with goat anti-human Fc (Jackson immunoresearch 109-005-098) andanti-goat IRDYE800 (Li-cor 926-32214) before analysis on the Li-CorOdyssey infrared scanner. The % of mAb OUT as a function of time isshown in FIG. 24.

The results demonstrate that internalization for all mAbs is comparableand that none of the mAbs internalizes completely. Initially, between 0and 2 hours, the mAb internalization goes very fast, but then it seemsthat a steady state is reached where about 30% of the mAb remains at theoutside of the cell, even after 24 hours of incubation at 37° C.

The internalization of CD70-bound mAbs was also assessed using othercell lines. In these experiments, only the signal of the mAb outside thecell was measured as a function of time using FACS analysis according tothe protocol described below. This alternative protocol was determinedto be a reliable readout as compared with the method described above(see FIG. 24, right-hand panel).

For suspension cells, cells were centrifuged, counted and the pelletresuspended in medium to a density of 5×10⁵ cells/ml, and 1000 of thissuspension was added per well of a 96-well V-bottom plate. For adherentcells, cells were seeded in a 96-well plate and grown ON at 37° C., 5%CO₂ until cells were fully attached. Cells were plated at a densitywhich allowed linear cell growth for an additional 48 h period.

The mAbs for testing were diluted and incubated with the cells at 37°C., 5% CO₂ for varying time periods: 24 h, 8 h, 6 h, 4 h, 2 h, 1 h, 30min, 15 min, 5 min, 0 min (=no mAb). Following incubation, the plateswere washed on ice with cold FACS buffer to stop the internalizationreaction. At this stage, adherent cells were detached from the plateusing cell dissociation solution (Sigma) and transferred to a V-bottom96-well plate. Next, cells were spun down at 4° C. The supernatant wasremoved by gently inverting the plate. The cells were washed twice bygently re-suspending the cell pellet in 100 μl cold FACS buffer. Afterwashing, the cell pellet was resuspended in 100 μl anti-hu IgG-FITC,diluted 1/500 in FACS buffer. The plate was incubated for 30 min at 4°C., while shaking, and the cells were washed a further three times withcold FACS buffer. The cells were resuspended in 100 μl FACS buffer andfluorescence was measured immediately. The median mean fluorescenceintensity (MFI) was plotted versus time.

Several of the experiments were repeated two or more times. Results fromtwo experiments were very comparable. In some experiments, using U266,SU-DHL-6 and Raji cells, a concentration range (20 ng/ml-5 μg/ml) wasused to see if there was an effect of mAb concentration oninternalization rate. No such effect was observed (data not shown).

The results for different cell lines tested are shown in Table 29 anddemonstrate that internalization is not a common phenomenon, actually itis quite a rare event. Table 29 summarizes the percentage ofinternalization after 6 hours for the different cell lines. Theseresults show that most of ARGX-110 remains bound to the outside of thecells making the cells more susceptible to ADCC, CDC and ADCP.

TABLE 29 % internalization of CD70 mAbs on different cell lines after 6hours. % internalization Average after 6 % per Type Cell line hoursindication Burkitt lymphoma Raji 2 2 Large B cell lymphoma SU-DHL-6 3 3Hodgkin lymphoma L428 15 15 Non Hodgkin lymphoma MHHPREB1 19 19 MantleCell Lymphoma Mino 12 7 Granta 519 7 Rec-1 2 Chronic lymphocytic Mec1 37 leukemia JVM-2 8 JVM-3 11 Patient HR 5 Patient HR 7 Patient LR 8Cutaneous T cell HUT78 35 33 lymphoma HH 31 Multiple Myeloma U266 38 34AMO-1 36 RPMI8226 38 MM1.S 35 KMS12MB 49 Renal cell carcinoma 786-O 5854 Caki-1 37 A498 66 Astrocytoma U251 52 52 Gastric carcinoma MKN-45 0 0Lung carcinoma A549 0 3 EBC-1 6 Melanoma WM1205-Lu 45 44 WM3248 34 WM11552 Glioblastoma U87MG 10 21 U343 32 Ovarian carcinoma SKOV3 51 51Pancreatic carcinoma PANC-1 29 25 PANC-89 20

TABLE 30 nucleotide sequences encoding selected CD70 antibodies mAb41D12 Heavy chain gccgccaccATGGGCTGGTCCTGCATCATCCTGTTTCTGGTGGCCACSEQ ID NO: CGCCACAGGCGTCCACTCT GAGGTGCAGCTCGTGGAGTCTGGG 344GGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGTCTACTACATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTCGAGTGGGTCTCAGATATTAATAATGAAGGTGGTACTACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACTCTAAGAACAGCCTGTATCTGCAAATGAACAGCCTGCGCGCCGAGGACACGGCCGTGTACTACTGCGCGAGAGATGCCGGATATAGCAACCATGTACCCATCTTTGATTCTTGGGGCCAGGGGACCCTGGTCACTGTCTCCTCAGCCAGTACAAAAGGTCCAAGTGTGTTCCCTCTTGCTCCCTCATCCAAGAGTACCAGTGGAGGCACCGCCGCTCTTGGCTGCTTGGTTAAGGATTATTTCCCAGAGCCTGTCACTGTTTCATGGAACTCCGGCGCCTTGACATCTGGTGTGCATACCTTTCCAGCCGTGCTGCAGTCAAGTGGCCTCTACAGCCTCAGTAGCGTGGTCACTGTGCCCAGCAGCTCTCTCGGCACACAAACTTATATCTGTAATGTGAATCATAAGCCTTCAAATACCAAGGTGGATAAGAAAGTGGAACCAAAATCATGTGACAAGACACACACCTGCCCTCCTTGTCCAGCCCCCGAACTGCTGGGTGGGCCCAGCGTGTTCCTGTTTCCTCCTAAACCCAAAGACACTCTGATGATTAGTAGGACCCCAGAAGTCACTTGCGTGGTGGTTGACGTGTCACATGAAGATCCCGAGGTCAAGTTCAATTGGTATGTTGACGGGGTCGAAGTTCACAACGCTAAAACTAAACCAAGAGAGGAACAGTATAACTCTACCTACCGGGTGGTGAGTGTTCTGACTGTCCTCCATCAAGACTGGCTGAATGGCAAAGAATACAAGTGTAAGGTGAGCAACAAAGCCCTGCCCG CTCCTATAGAGAAAACAATATCCAAAGCCAAAGGTCAACCTCGCGAGCCACAGGTGTACACCCTCCCACCAAGCCGCGATGAACTTACTAAGAACCAAGTCTCTCTTACTTGCCTGGTTAAGGGGTTCTATCCATCCGACATTGCAGTCGAGTGGGAGTCTAATGGACAGCCTGAGAACAACTACAAAACCACCCCTCCTGTTCTGGATTCTGACGGATCTTTCTTCCTTTATTCTAAACTCACCGTGGATAAAAGCAGGTGGCAGCAGGGCAACGTGTTCAGCTGTTCCGTTATGCATGAGGCCCTGCATAACCATTATACCCAGAAGTCTTTGTCCCTCAGTCCAGGAAAGTG A kozak LEADERVARIABLE REGION LAMBDA CONSTANT REGION Light chaingccgccaccATGGGCTGGTCCTGCATCATCCTGTTTCTGGTGGCCAC SEQ ID NO:CGCCACAGGCGTCCACTCT CAGGCAGTGGTGACCCAGGAGCCT 345TCCCTGACAGTGTCTCCAGGAGGGACGGTCACGCTCACCTGCGGCCTCAAATCTGGGTCTGTCACTTCCGATAACTTCCCCACTTGGTACCAGCAGACACCAGGCCAGGCTCCCCGATTGCTTATCTACAACACAAACACCCGTCACTCTGGCGTCCCCGACCGCTTCTCCGGATCCATCCTGGGCAACAAAGCCGCCCTCACCATCACGGGGGCCCAGGCCGACGACGAGGCCGAATATTTCTGTGCTCTGTTCATAAGTAATCCTAGTGTTGAGTTCGGCGGAGGGACCCAACTGACCGTCCTAGGTCAACCTAAAGCAGCACCTTCAGTTACTCTGTTTCCACCTAGTTCAGAGGAACTGCAGGCCAATAAAGCCACACTCGTCTGCCTCATCAGTGACTTCTACCCAGGAGCCGTGACCGTGGCCTGGAAAGCCGACAGTAGCCCCGTGAAGGCCGGGGTGGAGACAACAACTCCTAGTAAACAGAGTAATAACAAATATGCCGCTAGTAGTTATCTCTCCCTCACTCCCGAGCAGTGGAAGTCTCACAGAAGTTACTCTTGTCAGGTTACTCACGAGGGTTCCACAGTGGAAAAGACTGTGGCCCCTACTGAA TGTAGTTGA kozak LEADERVARIABLE REGION LAMBDA CONSTANT REGION 57B6 Heavy chainCAGTTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTG SEQ ID NO: GGGGGTCTCTGAGACTC346 TCTTGTGCAGCCTCTGGATTCAGCTTCAGTCACTATGCCATGAGC TGGGTCCGCCAGGCTCCAGGAAAGGGGCTAGAGTGGGTCTCAGGTGATAATACCTACGA TGGTGGTACAAGGTATCAAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGG CAAGAACACGCTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTGTATTA CTGTGCAAAAGATACTGGTAGAGGCATTATGGGGGAGTACGGCATGGACTACTGGGGCAA AGGGACCCTGGTCACC GTCTCCTCALight chain CAGACTGTGGTGACCCAGGAGCCGTCCCTGTCAGTGTCTCCAGG SEQ ID NO:AGGGACGGTCACACTC 347 ACCTGCGGCCTCAAGTCTGGGTCTGTCACTTCCAGTAACTACCCTGCTTGGTACCAGCAG ACACCAGGCCAGGCTCCCCGATTGCTTATCTACAACACAAACAGCCGTCACTCTGGGGTC CCCAGTCGCTTCTCCGGATCCATCTCTGGGAACAAAGCCGCCCTCACCATCACGGGGGCC CAGCCCGAGGACGAGGCCGACTATTACTGTGCTCTGTACATGGGTAGTGGTAGTGCCAAT GCTATGTTCGGCGGAGGGACCCATCTGACCGTCCTGGGTCA 59D10Heavy chain GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTG SEQ ID NO:GGGGGTCTCTGAGACTC 348 TCCTGTGCAGCGTCCGAATTGTCCTTCAGTATTTCTGAGATGACCTGGGTCCGCCAGGCT CCAGGAAAGGGGCTCGAGTGGGTCTCAGGTATTAGTGGTGTAACTGGTGGTAGTAGTACA AGTTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGACAAGAACACG TTGTATCTACAAATGAACAGCCTGATACCCGAGGACACGGCCGTATATTACTGTGCAACA ACTAGTGGTACTTACTACTTCATCCCCGAGTATGAGTACTGGGGCCAGGGGACCCAGGTC ACCGTCTCCTCA Light chainCAGTCTGTGCTGACCCAGCCTCCCTCCGTGTCTGGGTCTCCAGG SEQ ID NO: AAAGACGGTCACCATC349 TCCTGTGCAGGAACCAGCAGTGATGTTGGGTATGGATACTATGTC TCCTGGTATCAACAGTTCCCAGGAATGGCCCCCAAACTCCTGATATATGACGTCAATAAA CGGGCCTCAGGGATCGCTGATCGCTTCTCTGGCTCCAAGGCCGGCAACACTGCCTCCCT GACCATCTCTGGGCTCCAGTCTGAGGACGAGGCTGATTATTACTGTGCCTCATATAGAAGT AGCGCCAATGCTGTGTTCGGCGGAGGGACCCATCTGACCGTCCTGGGT

The invention claimed is:
 1. A monoclonal antibody, or a fragmentthereof, which specifically binds to an epitope of human CD70, whereinthe epitope is within the amino acid sequence HIQVTLAICSS (SEQ IDNO:342), and which, when tested as a Fab fragment, has an off-rate(k_(off)) as measured by surface plasmon resonance for human CD70 ofless than 7×10⁻⁴ s⁻¹.
 2. The monoclonal antibody or fragment thereofaccording to claim 1, which, when tested as a Fab fragment, has anoff-rate (k_(off)) as measured by surface plasmon resonance for humanCD70 of less than or equal to 4.8×10⁻⁴ s⁻¹.
 3. The monoclonal antibodyor fragment thereof according to claim 1, which is a chimeric antibodyor a humanized antibody.
 4. The monoclonal antibody or fragment thereofaccording to claim 1, which is a chimeric antibody, wherein the chimericantibody comprises a constant domain derived from a humanimmunoglobulin.
 5. The chimeric antibody according to claim 4, whereinthe constant domain is derived from a human immunoglobulin selected fromthe group consisting of IgG1, IgG2, IgG3, and IgG4.
 6. The chimericantibody according to claim 4, wherein the constant domain is derivedfrom human IgG1.
 7. The monoclonal antibody or fragment thereofaccording to claim 1, wherein the monoclonal antibody or fragmentthereof comprises a heavy chain variable (VH) domain and a light chainvariable (VL) domain, wherein the VH and VL domains, or one or morecomplementarity-determining regions (CDRs) thereof, are derived from acamelid species.
 8. The monoclonal antibody or fragment thereof claim 1,wherein the monoclonal antibody or fragment thereof is a llama-humanchimeric antibody comprising VH and VL domains, or one or more CDRsthereof, derived from llama, and one or more constant domains derivedfrom a human immunoglobulin.
 9. The monoclonal antibody or fragmentthereof according to claim 1, which is capable of directing immuneeffector function against a cell expressing human CD70 on its surface,wherein the immune effector function is selected from the groupconsisting of antibody-dependent cellular cytotoxicity (ADCC),complement-dependent cytotoxicity (CDC), antibody-dependent cellularphagocytosis (ADCP), and any combination thereof.
 10. The monoclonalantibody or fragment thereof according to claim 1, wherein the antibodyfragment is an scFv, a F(ab′)₂ fragment, a Fab fragment, a Fab′fragment, an Fd fragment, an Fv fragment, or a single domain antibody(Dab) fragment.
 11. A pharmaceutical composition comprising themonoclonal antibody or fragment thereof according to claim 1 and apharmaceutically acceptable carrier or excipient.
 12. An immunoconjugatecomprising the monoclonal antibody or fragment thereof according toclaim 1 and a cytotoxic agent.
 13. The immunoconjugate according toclaim 12, wherein the cytotoxic agent is selected from the groupconsisting of chemotherapeutic agents, toxins of bacterial, fungal,plant or animal origin, and radioactive isotopes.